Rafael Franco - Academia.edu (original) (raw)

Papers by Rafael Franco

Neuropsychopharmacology, 2000

Evidence has been obtained for adenosine/dopamine interactions in the central nervous system. The... more 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 subtypespecific 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

Trends in Neurosciences, 2007

The existence of neurotransmitter receptor heteromers is becoming broadly accepted and their func... more The existence of neurotransmitter receptor heteromers is becoming broadly accepted and their functional significance is being revealed. Heteromerization of neurotransmitter receptors produces functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer. Neurotransmitter receptor heteromers can function as processors of computations that modulate cell signaling. Thus, the quantitative or qualitative aspects of the signaling generated by stimulation of any of the individual receptor units in the heteromer are different from those obtained during coactivation. Furthermore, recent studies demonstrate that some neurotransmitter receptor heteromers can exert an effect as processors of computations that directly modulate both pre-and postsynaptic neurotransmission. This is illustrated by the analysis of striatal receptor heteromers that control striatal glutamatergic neurotransmission.

$Research paper thumbnail of Heterogeneous localization of some purine enzymes in subcellular fractions of rat brain and cerebellum$

Neurochemical Research, 1986

The activity of guanine deaminase (GAH, E.C. 3.5.4.3) was lower in rat cerebellum soluble and mic... more The activity of guanine deaminase (GAH, E.C. 3.5.4.3) was lower in rat cerebellum soluble and microsomal fractions than in rat brain subfractions. Adenosine deaminase (ADA, E.C. 3.5.4.4) activity was released in higher proportion than guanine deaminase, purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4), 5′-nucleotidase (5′N, E.C. 3.1.3.5), and lactate (LDH, E.C. 1.1.1.27) and malate (MDH, E.C. 1.1.1.37) dehydrogenase in press-juices of rat brain. Furthermore, nerve ending-derived fractions (synaptosomes and synaptic vesicles) showed an enrichment of adenosine deaminase and also of 5′-nucleotidase. The action of deoxycholate over the subfractions did not increase the activity of either enzyme. The contrary occurred with the remaining enzymes studied. Thus, it is possible that one set of enzymes are located on the surface of the particulate vesicles, whereas another set are located inside these vesicles, suggesting a compartmentation of purine catabolic enzymes in different areas of the central nervous system.

Trends in Biochemical Sciences, 2003

G-protein-coupled receptors form homomers and heteromers; agonist-induced conformational changes ... more G-protein-coupled receptors form homomers and heteromers; agonist-induced conformational changes within interacting receptors of the oligomer modify their pharmacology, signalling and/or trafficking. When these receptors are activated, the oligomers rearrange and cluster and a novel mechanism of receptoroperation regulation by oligomer intercommunication is possible. This intercommunication would be assisted by components of the plasma membrane and by scaffolding proteins. Receptor cross-sensitization, crossdesensitization and novel, integrated receptor responses can then develop between oligomeric receptor complexes of the cluster without direct contact between them. This concept gives a new perspective to the understanding of neurotransmission and neuronal plasticity.

European Journal of Biochemistry, 1984

A computer model of purine metabolism, including catabolism, salvage pathways and interconversion... more A computer model of purine metabolism, including catabolism, salvage pathways and interconversion among nucleotides, is given. Steady-state rate equations corresponding to metabolic enzymes are written based on information from the literature about their kinetic behaviour. Numerical integration of this set of equations is performed employing selected parameters taken from the literature. After stabilization of purine compound concentrations is reached, simulation of enzyme deficit and enzyme overproduction is carried out. The latter is calculated by varying specified maximum velocities in the numerical integration. A pattern of intermediate metabolite concentrations is found. These results form a basis for the comparison of normal patterns or patterns reflecting the effects of inborn errors of metabolism.

Experimental Cell Research, 2003

The involvement of caveolae in the internalization of A(1) adenosine receptors (A(1)R) and the re... more The involvement of caveolae in the internalization of A(1) adenosine receptors (A(1)R) and the receptor sorting and recycling was studied in the smooth muscle cell line DDT(1)MF-2, by binding assays, by confocal microscopy, and at the structural level. The use of cholera toxin-binding subunit adsorbed to gold as a specific probe for labeling the ganglioside GM(1) and immunoelectron microscopy techniques showed that agonist stimulation produced a clustering and sequestration of adenosine receptors in caveolae. Furthermore, pull-down experiments showed there to be a direct interaction between the C-terminal domain of A(1)R and caveolin-1. Addition of exogenous adenosine deaminase (ADA), a protein that binds to A(1)R and acts as a receptor activity modifying protein (RAMP) stimulated R-PIA-induced A(1) receptor internalization. Finally, the sorting and recycling of A(1)R/ADA complexes was analyzed. Detailed electron microscopy revealed that A(1)R/ADA complexes internalize together through caveolae, are differentially sorted in endosomes, and are recycled back to the cell surface by different groups of recycling endosomes. These results give insight into the spatiotemporal regulation and traffic of A(1)R and RAMPs.

Molecular and Cellular Biology, 2000

A 1 adenosine receptors (A 1 Rs) are G protein-coupled heptaspanning receptors that interact at t... more 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 proteincoupled 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.

Parkinsonism & Related Disorders, 2004

BRET competition experiments were performed using a chimeric D 2 R-D 1 R in which helices 5 and 6... more BRET competition experiments were performed using a chimeric D 2 R-D 1 R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D 2 R were replaced by those of the dopamine D 1 receptor (D 1 R). Although the wild type D 2 R 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 D 2 R holds the site(s) for interaction with A 2A R.

Journal of Neuroscience Research, 1988

Rat brain adenosine deaminase (E. C. 3.5.4.4.) was purified 667-fold from the supernatant fractio... more Rat brain adenosine deaminase (E. C. 3.5.4.4.) was purified 667-fold from the supernatant fraction by the following techniques: heat treatment (60°C), fraction-ation with ammonium sulfate, column chromatography on DEAE-Sepharose, and preparative gel electrophoresis. The purified enzyme was homogeneous by the criterion of polyacrylamide disc gel electrophoresis and isoelectric focusing. Amino acid composition is given. The isoelectric point of the enzyme (5.2) was determined by isoelectric focusing on agarose. The apparent molecular weight was estimated to be 39,000 (Stokes Radius [Rs] = 27.3 Å) using a calibrated Sephacryl S-300 column. The study of the influence of the temperature on the initial reaction rates allowed calculation of Ea (8.9 Kcal/mole) and ΔH (5.0 Kcal/mole) values. The variation of V and Km with pH suggests the existence of a sulfhydryl group and an imidazole group in the enzyme-substrate complex. The enzyme had a Km (adenosine) of 4.5 × 10−5 M and was inhibited by inosine, guanosine, adenine, and hypoxanthine but not by other intermediates of purine metabolism. None of the inhibitors were active as substrates. The enzyme was also inhibited by dimethyl sulfoxide and ethanol. Inhibition by ethanol can account partially for the CNS depressant effects of levels 3 and 4 of alcohol intoxication. A number of drugs having therapeutic uses such as sedative, anxiolytic, analgesic, and relaxant are modulators of the enzyme. Among these, lidoflazine, phenylbutazone, and chlordiazepoxide are the most potent as inhibitors (Ki 30, 54, and 83 μM, respectively), whereas medazepam is the most potent as activator (Ka 0.32 mM). Thus, it is concluded that some drugs that inhibit adenosine uptake also modulate adenosine deaminase activity. Besides, since the enzyme is located extracellularly [Franco et al, 1986], these drugs can modulate the physiological effects exerted by extracellular adenosine.

Current Pharmaceutical Design, 2008

Adenosine A 2A -dopamine D 2 receptor interactions play a very important role in striatal functio... more Adenosine A 2A -dopamine D 2 receptor interactions play a very important role in striatal function. A 2A -D 2 receptor interactions provide an example of the capabilities of information processing by just two different G protein-coupled receptors. Thus, there is evidence for the coexistence of two reciprocal antagonistic interactions between A 2A and D 2 receptors in the same neurons, the GABAergic enkephalinergic nens. An antagonistic A 2A -D 2 intramembrane receptor interaction, which depends on A 2A -D 2 receptor heteromerization and G q/11 -PLC signaling, modulates neuronal excitability and neurotransmitter release. On the other hand, an antagonistic A 2A -D 2 receptor interaction at the adenylyl-cyclase level, which depends on G s/olf -and G i/o -type V adenylyl-cyclase signaling, modulates protein phosphorylation and gene expression. Finally, under conditions of upregulation of an activator of G protein signaling (AGS3), such as during chronic treatment with addictive drugs, a synergistic A 2A -D 2 receptor interaction can also be demonstrated. AGS3 facilitates a synergistic interaction between G s/olf -and G i/o -coupled receptors on the activation of types II/IV adenylyl cyclase, leading to a paradoxical increase in protein phosphorylation and gene expression upon co-activation of A 2A and D 2 receptors. The analysis of A 2 -D 2 receptor interactions will have implications for the pathophysiology and treatment of basal ganglia disorders and drug addiction.

Journal of Neuroscience Research, 1995

The A1 adenosine receptor from pig brain cortex has been identified by means of two antipeptide a... more The A1 adenosine receptor from pig brain cortex has been identified by means of two antipeptide antibodies against two domains of the receptor molecule: PC/10 antiserum was raised against a part of the third intracellular loop, and PC/20 antiserum was raised against a part of the second extracellular loop. PC/10 antibody was able to recognize a 39-kDa band that corresponded to the Al receptor, as demonstrated by immunoblotting and by immunoprecipitation of the molecule cross-linked to [125I](R)-2-azidoN2-p-hydroxy(phenylisopropyl)adenosine. Besides the 39-kDa band, PC/20 also recognized a 74-kDa form that does not seem to correspond to a receptor-G protein complex. The occurrence of the two bands was detected and analyzed in samples from different species and tissues showing a heterogeneous distribution of both. The 74-kDa form can be converted into the 39-kDa form by treatment with agonists or antagonists of Al adenosine receptors. These results suggest that A1 adenosine receptor can occur in dimers and that the dimer–monomer conversion might be regulated by adenosine as the physiological ligand. Since the 74-kDa aggregates were not recognized by PC/10, it is likely that part of the third intracellular loop participates in the protein–protein interaction. ©1995 Wiley-Liss, Inc.

Proceedings of The National Academy of Sciences, 2002

The physiological meaning of the coexpression of adenosine A2A receptors and group I metabotropic... more The physiological meaning of the coexpression of adenosine A2A receptors and group I metabotropic glutamate receptors in ␥aminobutyric acid (GABA)ergic striatal neurons is intriguing. Here we provide in vitro and in vivo evidence for a synergism between adenosine and glutamate based on subtype 5 metabotropic glutamate (mGluR5) and adenosine A2A (A2AR) receptor͞receptor interactions.

Immunological Reviews, 1998

Summary: Adenosine deaminase (ADA, EC 3.5.4.4) is an enzyme of the purine metabolism which has be... more Summary: Adenosine deaminase (ADA, EC 3.5.4.4) is an enzyme of the purine metabolism which has been the object of considerable interest mainly because the congenital defect causes severe combined immunodeficiency (SCID). In the last 10 years, ADA, which was considered to be cytosolic, has been found on the cell surface of many ceils and, therefore, it can be considered an ecto-enzyme. There is recent evidence about a specific role of ecto-ADA, which is different from bat of intracellular ADA. Apart from degrading extracellular adenosine (Ado) or 2′-deoxyadenosLne (dAdo), which are toxic for lymphocytes, ecto-ADA has an extraenzymatic function via its interaction with GD26. ADA/CD26 interaction results in co-stimulatory signals in T cells. This co-stimulation is blocked by HIV-1, thus evidencing a role for ecto-ADA in the pathophysiology of AIDS. The fact that, besides CD26, ADA can interact with different cell-surface proteins opens new perspectives in the research for a role of ecto-ADA in the function of the immune system and in the Interactions that take place between different cells in the development of the immune system. The most interesting aspect is the possible participation of the ecto-enzyme in cell-to-cell contacts during ontogenesis and maturation of immunocompetent cells.

Proceedings of The National Academy of Sciences, 2005

Adenosine deaminase (ADA), a protein whose deficit leads to severe combined immunodeficiency, bin... more 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.

Febs Letters, 1996

Adenosine deaminase (ADA) is not only a cytosolic enzyme but can be found as an ecto-enzyme. At t... more 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 ADMCD26 complex is unclear. Here by confocal microscopy, affinity chromatography and coprecipitation experiments we show that A 1 adenosine receptor (AIR) is a second ecto-ADA binding protein. Binding of ADA to AtR increased its affinity for the ligand thus suggesting that ADA was needed for an effective coupling between A~R 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 AtR. 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 AIR.

Journal of Neurochemistry, 2002

Abstract: Adenosine deaminase is an enzyme of purine metabolism that has largely been considered ... more Abstract: Adenosine deaminase is an enzyme of purine metabolism that has largely been considered to be cytosolic. A few years ago, adenosine deaminase was reported to appear on the surface of cells. Recently, it has been demonstrated that adenosine deaminase interacts with a type II membrane protein known as either CD26 or dipeptidylpeptidase IV. In this study, by immunoprecipitation and affinity chromatography it is shown that adenosine deaminase and A1 adenosine receptors interact in pig brain cortical membranes. This is the first report in brain demonstrating an interaction between a degradative ectoenzyme and the receptor whose ligand is the enzyme substrate. By means of this interaction adenosine deaminase leads to the appearance of the high-affinity site of the receptor, which corresponds to the receptor-G protein complex. Thus, it seems that adenosine deaminase is necessary for coupling A1 adenosine receptors to heterotrimeric G proteins.

Recently evidence has been presented that adenosine A 2A and dopamine D 2 receptors form function... more Recently evidence has been presented that adenosine A 2A and dopamine D 2 receptors form functional heteromeric receptor complexes as demonstrated in human neuroblastoma cells and mouse fibroblast Ltk Ϫ cells. These A 2A /D 2 heteromeric receptor complexes undergo coaggregation, cointernalization, and codesensitization on D 2 or A 2A receptor agonist treatments and especially after combined agonist treatment. It is hypothesized that the A 2A /D 2 receptor heteromer represents the molecular basis for the antagonistic A 2A /D 2 receptor interactions demonstrated at the biochemical and behavioral levels. Functional heteromeric complexes between A 2A and metabotropic glutamate 5 receptors (mGluR5) have also recently been demonstrated in HEK-293 cells and rat striatal membrane preparations. The A 2A /mGluR5 receptor heteromer may account for the synergism found after combined agonist treatments demonstrated in different in vitro and in vivo models. D 2 , A 2A , and mGluR5 receptors are found together in the dendritic spines of the striatopallidal GABA neurons. Therefore, possible D 2 /A 2A /mGluR5 multimeric receptor complexes and the receptor interactions within them may have a major role in controlling the dorsal and ventral striatopallidal GABA neurons involved in Parkinson's disease and in schizophrenia and drug addiction, respectively.

Nature Chemical Biology, 2009

Journal of Molecular Neuroscience, 2005

The existence of A2A-D2 heteromeric complexes is based on coimmunoprecipitation studies and on fl... more The existence of A2A-D2 heteromeric complexes is based on coimmunoprecipitation studies and on fluorescence resonance energy transfer and bioluminescence resonance energy transfer analyses. It has now become possible to show that A2A and D2 receptors also coimmunoprecipitate in striatal tissue, giving evidence for the existence of A2A-D2 heteromeric receptor complexes also in rat striatal tissue. The analysis gives evidence that these heteromers are constitutive, as they are observed in the absence of A2A and D2 agonists. The A2A-D2 heteromers could either be A2A-D2 heterodimers and/or higher-order A2A-D2 hetero-oligomers. In striatal neurons there are probably A2A-D2 heteromeric complexes, together with A2A-D2 homomeric complexes in the neuronal surface membrane. Their stoichiometry in various microdomains will have a major role in determining A2A and D2 signaling in the striatopallidal GABA neurons. Through the use of D2/D1 chimeras, evidence has been obtained that the fifth transmembrane (TM) domain and/or the 13 of the D2 receptor are part of the A2A-D2 receptor interface, where electrostatic epitope-epitope interactions involving the N-terminal part of 13 of the D2 receptor (arginine-rich epitope) play a major role, interacting with the carboxyl terminus of the A2A receptor. Computerized modeling of A2A-D2 heteromers are in line with these findings. It seems likely that A2A receptor-induced reduction of D2 receptor recognition, G protein coupling, and signaling, as well as the existence of A2A-D2 co-trafficking, are the consequence of the existence of an A2A-D2 receptor heteromer. The relevance of A2A-D2 heteromeric receptor complexes for Parkinson’s disease and schizophrenia is emphasized as well as for the treatment of these diseases. Finally, recent evidence for the existence of antagonistic A2A-D3 heteromeric receptor complexes in cotransfected cell lines has been summarized.

Molecular and Cellular Biochemistry, 1995

A mathematical model based on kinetic data taken from the literature is presented for the pentose... more A mathematical model based on kinetic data taken from the literature is presented for the pentose phosphate pathway in fasted rat liver steady-state. Since the oxidative and non oxidative pentose phosphate pathway can act independently, the complete (oxidative + non oxidative) and the non oxidative pentose pathway were simulated. Sensitivity analyses are reported which show that the fluxes are mainly regulated by D-glucose-6-phosphate dehydrogenase (for the oxidative pathway) and by transketolase (for the non oxidative pathway). The most influent metabolites were the group ATP, ADP, P1 and the group NADPH, NADP+ (for the non oxidative pathway).