RESUMEN
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.
Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad/genética , Multimerización de Proteína , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D4/metabolismo , Animales , Células CHO , Cuerpo Estriado/metabolismo , Cricetinae , Técnicas de Sustitución del Gen/métodos , Ácido Glutámico/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/metabolismo , Transducción de Señal , Transfección/métodosRESUMEN
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.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Línea Celular , Membrana Celular/metabolismo , ADN Complementario , Colorantes Fluorescentes/metabolismo , Humanos , Inmunohistoquímica , Microscopía Confocal , Fosforilación , Receptores Acoplados a Proteínas G/genéticaRESUMEN
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 neurons. 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.
Asunto(s)
Receptor de Adenosina A2A/fisiología , Receptores de Dopamina D2/fisiología , Agonistas del Receptor de Adenosina A2 , Antagonistas del Receptor de Adenosina A2 , Adenilil Ciclasas/metabolismo , Animales , Ganglios Basales/fisiología , Enfermedades de los Ganglios Basales/tratamiento farmacológico , Enfermedades de los Ganglios Basales/fisiopatología , Antagonistas de los Receptores de Dopamina D2 , Encefalinas/metabolismo , Activación Enzimática , Proteínas de Unión al GTP/fisiología , Humanos , Neuronas/metabolismo , Fosforilación , Receptores de Dopamina D2/agonistas , Trastornos Relacionados con Sustancias/tratamiento farmacológico , Trastornos Relacionados con Sustancias/fisiopatología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
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.
Asunto(s)
Receptores Acoplados a Proteínas G/efectos de los fármacos , Algoritmos , Animales , Humanos , Ligandos , Modelos Químicos , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genéticaRESUMEN
In 1980/81 Agnati and Fuxe introduced the concept of intramembrane receptor-receptor interactions and presented the first experimental observations for their existence in crude membrane preparations. The second step was their introduction of the receptor mosaic hypothesis of the engram in 1982. The third step was their proposal that the existence of intramembrane receptor-receptor interactions made possible the integration of synaptic (WT) and extrasynaptic (VT) signals. With the discovery of the intramembrane receptor-receptor interactions with the likely formation of receptor aggregates of multiple receptors, so called receptor mosaics, the entire decoding process becomes a branched process already at the receptor level in the surface membrane. Recent developments indicate the relevance of cooperativity in intramembrane receptor-receptor interactions namely the presence of regulated cooperativity via receptor-receptor interactions in receptor mosaics (RM) built up of the same type of receptor (homo-oligomers) or of subtypes of the same receptor (RM type1). The receptor-receptor interactions will to a large extent determine the various conformational states of the receptors and their operation will be dependent on the receptor composition (stoichiometry), the spatial organization (topography) and order of receptor activation in the RM. The biochemical and functional integrative implications of the receptor-receptor interactions are outlined and long-lived heteromeric receptor complexes with frozen RM in various nerve cell systems may play an essential role in learning, memory and retrieval processes. Intramembrane receptor-receptor interactions in the brain have given rise to novel strategies for treatment of Parkinson's disease (A2A and mGluR5 receptor antagonists), schizophrenia (A2A and mGluR5 agonists) and depression (galanin receptor antagonists). The A2A/D2, A2A/D3 and A2A/mGluR5 heteromers and heteromeric complexes with their possible participation in different types of RM are described in detail, especially in the cortico-striatal glutamate synapse and its extrasynaptic components, together with a postulated existence of A2A/D4 heteromers. Finally, the impact of intramembrane receptor-receptor interactions in molecular medicine is discussed outside the brain with focus on the endocrine, the cardiovascular and the immune systems.
Asunto(s)
Encéfalo/fisiología , Membrana Celular/fisiología , Neuronas/fisiología , Receptor Cross-Talk/fisiología , Receptores de Neurotransmisores/fisiología , Transducción de Señal/fisiología , Animales , Membrana Celular/química , Membrana Celular/ultraestructura , Humanos , Neuronas/química , Neuronas/ultraestructura , Neurotransmisores/fisiología , Subunidades de Proteína/química , Subunidades de Proteína/fisiología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/fisiología , Receptores de Neurotransmisores/químicaRESUMEN
The molecular basis for the known intramembrane receptor-receptor interactions among heptahelical receptors (G protein coupled receptors, GPCR) was postulated to be heteromerization based on receptor subtype specific interactions between different types of homomers of GPCR. Adenosine and dopamine receptors in the basal ganglia have been fundamental to demonstrate the existence of receptor heteromers and the functional consequences of such molecular interactions. The heterodimer is only one type of heteromeric complex and the evidence is equally compatible with the existence of higher order heteromeric complexes, where also adapter proteins such as homer proteins and scaffolding proteins can exist, assisting in the process of linking the GPCR and ion channel receptors together in a receptor mosaic that may have special integrative value and may constitute the molecular basis for learning and memory. Heteromerization of D(2) dopamine and A(2A) adenosine receptors is reviewed by Fuxe in another article in this special issue. Here, heteromerization between D(1) dopamine and A(1) adenosine receptors is reviewed. Heteromers formed by dopamine D(1) and D(2) receptors and by adenosine A(1) and A(2A) receptors also occur in striatal cells and open new perspectives to understand why two receptors with apparently opposite effects are expressed in the same neuron and in the nerve terminals. The role of accessory proteins also capable of interacting with receptor-receptor heteromers in regulating the traffic and the molecular physiology of these receptors is also discussed. Overall, the knowledge of the reason why such complex networks of receptor-receptor and receptor-protein interactions occur in striatal cells is crucial to develop new strategies to combat neurological and neuropsychiatric diseases.
Asunto(s)
Membrana Celular/fisiología , Cuerpo Estriado/fisiología , Neuronas/fisiología , Receptor Cross-Talk/fisiología , Receptor de Adenosina A1/metabolismo , Receptores de Dopamina D1/metabolismo , Animales , Sitios de Unión/fisiología , Humanos , Sustancias Macromoleculares/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Since 1990 it has been known that dimers are the basic functional form of nearly all G-protein-coupled receptors (GPCRs) and that homo- and heterodimerization may play a key role in correct receptor maturation and trafficking to the plasma membrane. Nevertheless, homo- and heterodimerization of GPCR has become a matter of debate especially in the search for the precise physiological meaning of this phenomenon. This article focuses on how heterodimerization of adenosine A1 and A2A receptors, which are coupled to apparently opposite signalling pathways, allows adenosine to exert a fine-tuning modulation of striatal glutamatergic neurotransmission, providing a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release.
Asunto(s)
Neurotransmisores/metabolismo , Receptores Purinérgicos P1/metabolismo , Adenosina/metabolismo , Animales , Dimerización , HumanosRESUMEN
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 A(2B) 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 ADA-anchoring 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-gamma, TNF-alpha, and IL-6.
Asunto(s)
Adenosina Desaminasa/metabolismo , Células Dendríticas/inmunología , Dipeptidil Peptidasa 4/metabolismo , Glicoproteínas/metabolismo , Receptores Purinérgicos P1/metabolismo , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Adenosina Desaminasa/inmunología , Anticuerpos Monoclonales/inmunología , Cartilla de ADN , Dipeptidil Peptidasa 4/inmunología , Citometría de Flujo , Glicoproteínas/inmunología , Humanos , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Microscopía Confocal , Receptores Purinérgicos P1/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Recently evidence has been presented that adenosine A2A and dopamine D2 receptors form functional heteromeric receptor complexes as demonstrated in human neuroblastoma cells and mouse fibroblast Ltk- cells. These A2A/D2 heteromeric receptor complexes undergo coaggregation, cointernalization, and codesensitization on D2 or A2A receptor agonist treatments and especially after combined agonist treatment. It is hypothesized that the A2A/D2 receptor heteromer represents the molecular basis for the antagonistic A2A/D2 receptor interactions demonstrated at the biochemical and behavioral levels. Functional heteromeric complexes between A2A and metabotropic glutamate 5 receptors (mGluR5) have also recently been demonstrated in HEK-293 cells and rat striatal membrane preparations. The A2A/mGluR5 receptor heteromer may account for the synergism found after combined agonist treatments demonstrated in different in vitro and in vivo models. D2, A2A, and mGluR5 receptors are found together in the dendritic spines of the striatopallidal GABA neurons. Therefore, possible D2/A2A/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.
Asunto(s)
Cuerpo Estriado/metabolismo , Enfermedad de Parkinson/metabolismo , Receptor de Adenosina A2A/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular , Dimerización , Humanos , Sustancias Macromoleculares , Ratones , Enfermedad de Parkinson/terapia , Receptor del Glutamato Metabotropico 5 , Receptores de Dopamina D2/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Desaminasa/metabolismo , Adenosina/análogos & derivados , Corteza Cerebral/metabolismo , Fibroblastos/metabolismo , Neuronas/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores Purinérgicos P1/metabolismo , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Adenosina/metabolismo , Adenosina/farmacología , Adenosina Desaminasa/inmunología , Animales , Línea Celular , Corteza Cerebral/citología , Humanos , Inmunohistoquímica , Ratones , Microscopía Confocal , Agonistas del Receptor Purinérgico P1 , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/inmunología , Receptores Purinérgicos P1/inmunología , TransfecciónRESUMEN
OBJECTIVE: Previous epidemiologic studies have shown that around 5% of the population will suffer a tonic clonic seizure during their life. The aim of this study is to know how many and which of these people will suffer a second seizure and become epileptic. PATIENTS AND METHODS: 175 patients seen in the emergency department of the Vall d Hebron Hospital were included. They were divided in three groups according to the clinical suspicion of having had a seizure. Only the patients with low clinical suspicion and also normal EEG standard and EEG in sleep deprivation were excluded (16). The patients with previous episodes of lost of consciousness, previous episodes of possible mioclonias or absence were not excluded. RESULTS: After a first tonic clonic seizure the patients who did not receive treatment present a risk of relapse of 66% followed two years and the patients treated 46%. The difference between two groups was statistically significant. Dividing the patients according to the type of seizure: primary generalised, partial or nor localised we did not find differences in the risk of relapse. Dividing the patients according to their etiology we found that the group of patients with provoked seizures was different from the rest groups: symptomatic, genetic or cryptogenic and idiopathic, who had equal risk of recurrence. We found that the presence of previous episodes of lost of consciousness, the clinical suspicion and, probably (we obtained nearly statistical signification) de EEG and the presence of previous mioclonias or absences were risk factors. Other factor like age at the moment of the first episode, febrile seizures, familiar history, antecedents of stroke, encephalitis, neurosurgery and dementia were not related with the risk of relapse. CONCLUSIONS: With the exception of provoked seizures the rest of first tonic clonic seizures have a high risk of relapse (around 60 70%) and if they go with abnormal EEG, previous episodes of absences or mioclonias starting treatment must be considered.
Asunto(s)
Convulsiones/fisiopatología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Electroencefalografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mioclonía , Pronóstico , Recurrencia , Factores de Riesgo , Convulsiones/diagnóstico , Convulsiones/etiología , Convulsiones/terapiaRESUMEN
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.
Asunto(s)
Adenosina Desaminasa/inmunología , Citocinas/farmacología , Dipeptidil Peptidasa 4/inmunología , Activación de Linfocitos , Linfocitos T/inmunología , Adenosina Desaminasa/biosíntesis , Membrana Celular/enzimología , Membrana Celular/inmunología , Células Cultivadas , Citocinas/inmunología , Dipeptidil Peptidasa 4/biosíntesis , Humanos , Linfocitos T/enzimología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
Chronic exposure of A(1) adenosine receptors (A(1)R) to A(1)R agonists leads to activation, phosphorylation, desensitization, and internalization to intracellular compartments of the receptor. Desensitization and internalization of A(1)R is modulated by adenosine deaminase (ADA), an enzyme that regulates the extracellular concentration of adenosine. ADA interacts with A(1)R on the cell surface of the smooth muscle cell line DDT1 MF-2, and both proteins are internalized following agonist stimulation of the receptor. The mechanism involved in A(1)R and ADA internalization upon agonist exposure is poorly understood in epithelial cells. In this report, we show that A(1)R and ADA interact in LLC-PK(1) epithelial cells. Exposure of LLC-PK(1) cells to A(1)R agonists induces aggregation of A(1)R and ADA on the cell surface and their translocation to intracellular compartments. Biochemical and cell biology assays were used to characterize the intracellular vesicles containing both proteins after agonist treatment. A(1)R and ADA colocalized together with the rafts marker protein caveolin. Filipin, a sterol-binding agent that disrupts rafts (small microdomains of the plasma membrane), was able to inhibit A(1)R internalization. In contrast, acid treatment of the cells, which disrupts internalization via clathrin-coated vesicles, did not inhibit agonist-stimulated A(1)R internalization. We demonstrated that A(1)R agonist N(6)-(R)-phenylisopropyl adenosine promotes the translocation of A(1)R into low-density gradient fractions containing caveolin. Furthermore, a direct interaction of the C-terminal domain of A(1)R with caveolin-1 was demonstrated by pull down experiments. These results indicate that A(1)R and ADA form a stable complex in the cell surface of LLC-PK(1) cells and that agonist-induced internalization of the A(1) adenosine receptor and ADA is mediated by clathrin-independent endocytosis.
Asunto(s)
Adenosina Desaminasa/metabolismo , Caveolinas/fisiología , Células Epiteliales/metabolismo , Receptores Purinérgicos P1/metabolismo , Secuencia de Aminoácidos , Animales , Caveolina 1 , Células Cultivadas , Clatrina/metabolismo , Células Epiteliales/enzimología , Células Epiteliales/fisiología , Ligandos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Agonistas del Receptor Purinérgico P1 , PorcinosRESUMEN
We provide convergent and multiple evidence for a CD26/CXCR4 interaction. Thus, CD26 codistributes with CXCR4, and both coimmunoprecipitate from membranes of T (CD4(+)) and B (CD4(-)) cell lines. Upon induction with stromal cell-derived factor 1alpha (SDF-1alpha), CD26 is cointernalized with CXCR4. CXCR4-mediated down-regulation of CD26 is not induced by antagonists or human immunodeficiency virus (HIV)-1 gp120. SDF-1alpha-mediated down-regulation of CD26 is not blocked by pertussis toxin but does not occur in cells expressing mutant CXCR4 receptors unable to internalize. Codistribution and cointernalization also occurs in peripheral blood lymphocytes. Since CD26 is a cell surface endopeptidase that has the capacity to cleave SDF-1alpha, the CXCR4.CD26 complex is likely a functional unit in which CD26 may directly modulate SDF-1alpha-induced chemotaxis and antiviral capacity. CD26 anchors adenosine deaminase (ADA) to the lymphocyte cell surface, and this interaction is blocked by HIV-1 gp120. Here we demonstrate that gp120 interacts with CD26 and that gp120-mediated disruption of ADA/CD26 interaction is a consequence of a first interaction of gp120 with a domain different from the ADA binding site. SDF-1alpha and gp120 induce the appearance of pseudopodia in which CD26 and CXCR4 colocalize and in which ADA is not present. The physical association of CXCR4 and CD26, direct or part of a supramolecular structure, suggests a role on the function of the immune system and the pathophysiology of HIV infection.
Asunto(s)
Dipeptidil Peptidasa 4/metabolismo , Linfocitos/metabolismo , Receptores CXCR4/metabolismo , Adenosina Desaminasa/metabolismo , Sitios de Unión , Western Blotting , Antígenos CD4/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Regulación hacia Abajo , Endopeptidasas/metabolismo , Citometría de Flujo , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Células Jurkat , Mutación , Toxina del Pertussis , Pruebas de Precipitina , Unión Proteica , Seudópodos/metabolismo , Transfección , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Recently, evidence has emerged that seven transmembrane G protein-coupled receptors may be present as homo- and heteromers in the plasma membrane. Here we describe a new molecular and functional interaction between two functionally unrelated types of G protein-coupled receptors, namely the metabotropic glutamate type 1alpha (mGlu(1alpha) receptor) and the adenosine A1 receptors in cerebellum, primary cortical neurons, and heterologous transfected cells. Co-immunoprecipitation experiments showed a close and subtype-specific interaction between mGlu(1alpha) and A1 receptors in both rat cerebellar synaptosomes and co-transfected HEK-293 cells. By using transiently transfected HEK-293 cells a synergy between mGlu(1alpha) and A1 receptors in receptor-evoked [Ca(2+)](i) signaling has been shown. In primary cultures of cortical neurons we observed a high degree of co-localization of the two receptors, and excitotoxicity experiments in these cultures also indicate that mGlu(1alpha) and A1 receptors are functionally related. Our results provide a molecular basis for adenosine/glutamate receptors cross-talk and open new perspectives for the development of novel agents to treat neuropsychiatric disorders in which abnormal glutamatergic neurotransmission is involved.
Asunto(s)
Receptores de Glutamato Metabotrópico/metabolismo , Receptores Purinérgicos P1/metabolismo , Transducción de Señal , Animales , Línea Celular , Cerebelo/metabolismo , Humanos , Ratas , Receptor Cross-Talk , Sinaptosomas/metabolismoRESUMEN
Adenosine deaminase (ADA) is an enzyme of the purine metabolism that has been largely considered to be cytosolic. Recently, it has been demonstrated that the enzyme appears on the surface of lymphocytes where it interacts with the T-cell activation antigen CD26. ADA also appears on the surface of nonlymphoid cells anchored to adenosine A1 receptors. Here it is demonstrated that cell surface ADA in ADA+/CD26- T lymphocytes anchors to adenosine receptors of the A2B subtype (A2BR). An interaction between A2BR and cell surface ADA has been demonstrated in transfected Chinese hamster ovary cells and Jurkat J32 T lymphocytes. This has been proved by coimmunoprecipitation, binding of exogenous ADA to A2BR+ cells, and coimmunolocalization. The specificity of the interaction has also been demonstrated by the lack of interaction with other members of the G protein-coupled receptor superfamily. Binding of ADA to A2BR increases the affinity of the agonist 5'-N-ethylcarboxamidoadenosine and cAMP production. This effect occurs even when ADA devoid of enzyme activity is used. Therefore, in lymphocytes, cell surface ADA, apart from degrading extracellular adenosine, regulates those actions of adenosine that are mediated via adenosine receptors of the A2B subtype.
Asunto(s)
Adenosina Desaminasa/metabolismo , Receptores Purinérgicos P1/metabolismo , Linfocitos T/metabolismo , Adenosina Desaminasa/biosíntesis , Adenosina-5'-(N-etilcarboxamida)/farmacología , Animales , Sitios de Unión , Células CHO , Células Cultivadas , Cricetinae , AMP Cíclico/metabolismo , Humanos , Células Jurkat , Microscopía Confocal , Pruebas de Precipitina , Agonistas del Receptor Purinérgico P1 , Receptor de Adenosina A2B , Receptores Purinérgicos P1/genética , Sistemas de Mensajero Secundario/fisiología , Linfocitos T/enzimología , TransfecciónRESUMEN
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.
Asunto(s)
Receptores Dopaminérgicos/metabolismo , Receptores Purinérgicos P1/metabolismo , Animales , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Dimerización , Proteínas de Unión al GTP/metabolismo , Humanos , Modelos Biológicos , Receptores Dopaminérgicos/química , Receptores Dopaminérgicos/efectos de los fármacos , Receptores Purinérgicos P1/química , Receptores Purinérgicos P1/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
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)
Asunto(s)
Receptores Purinérgicos P1/metabolismo , Espermatozoides/metabolismo , Adenosina Desaminasa/metabolismo , Animales , Calcio/metabolismo , Radioisótopos de Calcio , Bovinos , Citometría de Flujo , Inmunohistoquímica , Técnicas In Vitro , Masculino , Microscopía Confocal , Permeabilidad , Agonistas del Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1RESUMEN
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.
Asunto(s)
Inhibidores de la Adenosina Desaminasa , Adenosina Desaminasa/metabolismo , Antígenos CD4/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Receptores CXCR4/metabolismo , Animales , Antígenos CD4/genética , Bovinos , Línea Celular , Dipeptidil Peptidasa 4/genética , Citometría de Flujo , Eliminación de Gen , VIH-1/fisiología , Humanos , Radioisótopos de Yodo , Ratones , Unión Proteica , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
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.