RESUMEN
The enzyme adenosine deaminase (ADA) is a multifunctional protein that can both degrade adenosine and bind extracellularly to adenosine receptors, acting as an allosteric modulator regulating the hormonal effects of adenosine. The molecular regions of ADA responsible for the latter are unknown. In this work, alanine scanning mutagenesis of various ADA amino acid stretches, selected through in silico docking experiments, allowed us to identify regions of the enzyme responsible for modulating both its catalytic activity and its ability to modulate agonist binding to A and A adenosine receptors (AR and AR). The combination of computational and in vitro experiments show that the structural gate to the catalytic site; i.e., the α-1 helix containing residues L58-I72 and the loop containing residues A184-I188 of ADA, were important to maintain both the catalytic efficiency of the enzyme and its action as an allosteric modulator of the adenosine receptors. These data are consistent with a predicted supramolecular assembly, in which ADA bridges AR and CD26 and are in line with the notion that the interaction of ADA with adenosine receptors has an important role in the immunosynapse. We propose that it is the ADA open form, but not the closed one, that is responsible for the functional interaction with A1R and A2AR.
Asunto(s)
Adenosina Desaminasa/química , Simulación del Acoplamiento Molecular , Receptor de Adenosina A1/química , Receptor de Adenosina A2A/química , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Regulación Alostérica/fisiología , Humanos , Mutagénesis Sitio-Dirigida , Mutación Missense , Unión Proteica , Estructura Secundaria de Proteína , Receptor de Adenosina A1/genética , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2A/metabolismoRESUMEN
Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.
Asunto(s)
Cuerpo Estriado/fisiopatología , Dominancia Cerebral/efectos de los fármacos , Discinesia Inducida por Medicamentos/fisiopatología , Levodopa/farmacología , Trastornos Parkinsonianos/fisiopatología , Receptores de Dopamina D1/fisiología , Receptores de Dopamina D3/fisiología , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Animales , Núcleo Caudado/efectos de los fármacos , Núcleo Caudado/fisiopatología , Cuerpo Estriado/efectos de los fármacos , Dimerización , Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Discinesia Inducida por Medicamentos/etiología , Regulación de la Expresión Génica/efectos de los fármacos , Levodopa/toxicidad , Macaca fascicularis , Masculino , Oxidopamina/toxicidad , Trastornos Parkinsonianos/inducido químicamente , Putamen/efectos de los fármacos , Putamen/fisiopatología , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/biosíntesis , Receptores de Dopamina D1/genética , Receptores de Dopamina D3/biosíntesis , Receptores de Dopamina D3/genéticaRESUMEN
Long-term therapy with L-3,4-dihydroxyphenylalanine (L-DOPA), still the most effective treatment in Parkinson's disease (PD), is associated with severe motor complications such as dyskinesia. Experimental and clinical data have indicated that adenosine A2A receptor antagonists can provide symptomatic improvement by potentiating L-DOPA efficacy and minimizing its side effects. It is known that the G-protein-coupled adenosine A2A, cannabinoid CB1 and dopamine D2 receptors may interact and form functional A2A-CB1-D2 receptor heteromers in co-transfected cells as well as in rat striatum. These data suggest that treatment with a combination of drugs or a single compound selectively acting on A2A-CB1-D2 heteromers may represent an alternative therapeutic treatment of PD. We investigated the expression of A2A-CB1-D2 receptor heteromers in the striatum of both naïve and hemiparkinsonian rats (HPD-rats) bearing a unilateral 6-hydroxydopamine (6-OHDA) lesion, and assessed how receptor heteromer expression and biochemical properties were affected by L-DOPA treatment. Radioligand binding data showed that A2A-CB1-D2 receptor heteromers are present in the striatum of both naïve and HPD-rats. However, behavioral results indicated that the combined administration of A2A (MSX-3 or SCH58261) and CB1 (rimonabant) receptor antagonists, in the presence of L-DOPA does not produce a response different from administration of the A2A receptor antagonist alone. These behavioral results prompted identification of heteromers in L-DOPA-treated animals. Interestingly, the radioligand binding results in samples from lesioned animals suggest that the heteromer is lost following acute or chronic treatment with L-DOPA.
Asunto(s)
Antiparkinsonianos/farmacología , Cuerpo Estriado/metabolismo , Lateralidad Funcional/efectos de los fármacos , Levodopa/farmacología , Trastornos Parkinsonianos/patología , Receptor Cross-Talk/fisiología , Antagonistas del Receptor de Adenosina A2/farmacología , Animales , Antagonistas de Receptores de Cannabinoides/farmacología , Inhibidores de la Colinesterasa/toxicidad , Cuerpo Estriado/efectos de los fármacos , Modelos Animales de Enfermedad , Dopaminérgicos/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Lateralidad Funcional/fisiología , Masculino , Oxidopamina/toxicidad , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/fisiopatología , Piperidinas/farmacología , Unión Proteica/efectos de los fármacos , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , Receptor Cross-Talk/efectos de los fármacos , Rimonabant , Tacrina/toxicidad , Factores de Tiempo , Temblor/inducido químicamenteRESUMEN
The molecular basis of priming for L-DOPA-induced dyskinesias in Parkinson's disease (PD), which depends on the indirect pathway of motor control, is not known. In rodents, the indirect pathway contains striatopallidal GABAergic neurons that express heterotrimers composed of A(2A) adenosine, CB(1) cannabinoid and D(2) dopamine receptors that regulate dopaminergic neurotransmission. The present study was designed to investigate the expression of these heteromers in the striatum of a primate model of Parkinson's disease and to determine whether their expression and pharmacological properties are altered upon L-DOPA treatment. By using the recently developed in situ proximity ligation assay and by identification of a biochemical fingerprint, we discovered a regional distribution of A(2A)/CB(1) /D(2) receptor heteromers that predicts differential D(2)-mediated neurotransmission in the caudate-putamen of Macaca fascicularis. Whereas heteromers were abundant in the caudate nucleus of both naïve and MPTP-treated monkeys, L-DOPA treatment blunted the biochemical fingerprint and led to weak heteromer expression. These findings constitute the first evidence of altered receptor heteromer expression in pathological conditions and suggest that drugs targeting A(2A)-CB(1) -D(2) receptor heteromers may be successful to either normalize basal ganglia output or prevent L-DOPA-induced side effects.
Asunto(s)
Antiparkinsonianos/farmacología , Núcleo Caudado/efectos de los fármacos , Levodopa/farmacología , Receptor de Adenosina A2A/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptores de Dopamina D2/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Agonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Animales , Núcleo Caudado/metabolismo , Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Antagonistas de los Receptores de Dopamina D2 , Macaca fascicularis , Masculino , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/metabolismo , Putamen/efectos de los fármacos , Putamen/metabolismo , Receptor Cannabinoide CB1/agonistasRESUMEN
Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D2 receptors function, we present evidence of σ1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with σ1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the σ1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain.