RESUMEN
The adenosine A(2A) receptor (A(2A) R) is a potential drug target for the treatment of Parkinson's disease and other neurological disorders. In rodents, the therapeutic efficacy of A(2A) R modulation is improved by concomitant modulation of the metabotropic glutamate receptor 5 (mGluR5). To elucidate the anatomical substrate(s) through which these therapeutic benefits could be mediated, pre-embedding electron microscopy immunohistochemistry was used to conduct a detailed, quantitative ultrastructural analysis of A(2A) R localization in the primate basal ganglia and to assess the degree of A(2A) R/mGluR5 colocalization in the striatum. A(2A) R immunoreactivity was found at the highest levels in the striatum and external globus pallidus (GPe). However, the monkey, but not the rat, substantia nigra pars reticulata (SNr) also harbored a significant level of neuropil A(2A) R immunoreactivity. At the electron microscopic level, striatal A(2A) R labeling was most commonly localized in postsynaptic elements (58% ± 3% of labeled elements), whereas, in the GPe and SNr, the labeling was mainly presynaptic (71% ± 5%) or glial (27% ± 6%). In both striatal and pallidal structures, putative inhibitory and excitatory terminals displayed A(2A) R immunoreactivity. Striatal A(2A) R/mGluR5 colocalization was commonly found; 60-70% of A(2A) R-immunoreactive dendrites or spines in the monkey striatum coexpress mGluR5. These findings provide the first detailed account of the ultrastructural localization of A(2A) R in the primate basal ganglia and demonstrate that A(2A) R and mGluR5 are located to interact functionally in dendrites and spines of striatal neurons. Together, these data foster a deeper understanding of the substrates through which A(2A) R could regulate primate basal ganglia function and potentially mediate its therapeutic effects in parkinsonism.
Asunto(s)
Ganglios Basales/metabolismo , Ganglios Basales/ultraestructura , Cuerpo Estriado/metabolismo , Cuerpo Estriado/ultraestructura , Receptor de Adenosina A2A/ultraestructura , Receptores de Glutamato Metabotrópico/ultraestructura , Secuencia de Aminoácidos , Animales , Ganglios Basales/química , Cuerpo Estriado/química , Dendritas/genética , Dendritas/metabolismo , Dendritas/ultraestructura , Femenino , Globo Pálido/química , Globo Pálido/metabolismo , Globo Pálido/ultraestructura , Células HEK293 , Haplorrinos , Humanos , Macaca mulatta , Masculino , Datos de Secuencia Molecular , Neuronas/química , Neuronas/metabolismo , Neuronas/ultraestructura , Ratas , Ratas Sprague-Dawley , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2A/metabolismo , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismoRESUMEN
Many transmembrane receptors are regulated by associations with scaffold proteins containing PDZ domains, which interact with receptor carboxyl-termini to control receptor signaling, trafficking, and localization. We describe here approaches for detecting and characterizing interactions between receptors and PDZ scaffolds. These approaches include the construction and screening of proteomic arrays, blot overlays using fusion proteins, and co-immunoprecipitation studies to assess interactions in cells.
Asunto(s)
Dominios PDZ , Análisis por Matrices de Proteínas/métodos , Proteómica/métodos , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular , Humanos , Inmunoprecipitación/métodos , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of ß-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with ß-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Arrestinas/genética , Arrestinas/metabolismo , Corteza Cerebral/anomalías , Corteza Cerebral/metabolismo , Células HEK293 , Humanos , Mutación , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/genética , Ubiquitinación/genética , Arrestina beta 2 , beta-ArrestinasRESUMEN
BACKGROUND & AIMS: Lysophosphatidic acid (LPA) is a potent inducer of colon cancer and LPA receptor type 2 (LPA(2)) is overexpressed in colon tumors. LPA(2) interacts with membrane-associated guanylate kinase with inverted orientation-3 (MAGI-3) and the Na+/H+ exchanger regulatory factor 2 (NHERF-2), but the biological effects of these interactions are unknown. We investigated the roles of MAGI-3 and NHERF-2 in LPA(2)-mediated signaling in human colon cancer cells. METHODS: We overexpressed or knocked down MAGI-3 in HCT116 and SW480 cells. The effects of MAGI-3 and NHERF-2 in LPA-induced cell migration, invasion, inositol phosphate generation, and nuclear factor-κB activation were determined. Expression of MAGI-3 and NHERF-2 in human colon tumor tissues was analyzed using tissue microarray analysis. RESULTS: NHERF-2 promoted migration and invasion of colon cancer cells, whereas MAGI-3 inhibited these processes. MAGI-3 competed with NHERF-2 for binding to LPA(2) and phospholipase C-ß3. However, NHERF-2 and MAGI-3 reciprocally regulated LPA(2)-induced phospholipase C activity. MAGI-3 increased the interaction of LPA(2) with Gα(12), whereas NHERF-2 preferentially promoted interaction between LPA(2) and Gα(q). MAGI-3 decreased the tumorigenic capacity of LPA(2) by attenuating the activities of nuclear factor-κB and c-Jun N-terminal kinase. MAGI-3 and NHERF-2 were expressed differentially in colon adenocarcinomas, consistent with their opposing effects. CONCLUSIONS: LPA(2) is dynamically regulated by 2 distinct PDZ proteins via modulation of G-protein coupling and receptor signaling. MAGI-3 is a negative regulator of LPA(2) signaling.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias del Colon/metabolismo , Lisofosfolípidos/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/metabolismo , Animales , Movimiento Celular , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Genes APC , Células HCT116 , Humanos , Fosfatos de Inositol/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de la Membrana/genética , Ratones , FN-kappa B/metabolismo , Invasividad Neoplásica , Fosfolipasa C beta/metabolismo , Fosfoproteínas/genética , Interferencia de ARN , Receptores del Ácido Lisofosfatídico/genética , Intercambiadores de Sodio-Hidrógeno/genética , Factores de Tiempo , Análisis de Matrices Tisulares , TransfecciónRESUMEN
The astrocytic glutamate transporter GLAST (also known as EAAT1) is a key regulator of extracellular glutamate levels in many regions of vertebrate brains. To identify novel interacting partners that might regulate the localization and function of GLAST in astrocytes, we screened the transporter's C-terminus (GLAST-CT) against a proteomic array of 96 different PDZ domains. The GLAST-CT robustly and specifically interacted with PDZ domains from two related scaffolding proteins, the Na(+)/H(+) exchanger regulatory factors 1 and 2 (NHERF-1 and NHERF-2). Studies on cultured rat cortical astrocytes revealed that these cells are highly enriched in NHERF-2 relative to NHERF-1. Endogenous GLAST and NHERF-2 from cultured astrocytes were found to robustly co-immunoprecipitate, and further co-immunoprecipitation studies on mutant versions of GLAST expressed in transfected cells revealed the GLAST/NHERF-2 interaction to be dependent on the last amino acid of the GLAST-CT. Knockdown of endogenous NHERF-2 in astrocytes via siRNA treatment resulted in a significant reduction in GLAST activity, which corresponded to significantly reduced total expression of GLAST protein and reduced half-life of GLAST, as assessed in pulse-chase metabolic labeling studies. These findings reveal that NHERF-2 can interact with GLAST in astrocytes to enhance GLAST stability and activity.
Asunto(s)
Transportador 1 de Aminoácidos Excitadores/metabolismo , Dominios PDZ/fisiología , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Ácido Aspártico/farmacocinética , Astrocitos , Células Cultivadas , Embrión de Mamíferos , Transportador 1 de Aminoácidos Excitadores/genética , Regulación de la Expresión Génica/genética , Humanos , Inmunoprecipitación/métodos , Mutación Missense/genética , Neocórtex/citología , Dominios PDZ/efectos de los fármacos , Fosfoproteínas/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Estructura Terciaria de Proteína , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Intercambiadores de Sodio-Hidrógeno/genética , Transfección/métodos , Tritio/farmacocinéticaRESUMEN
G protein-coupled receptors (GPCRs) mediate physiological responses to various ligands, such as hormones, neurotransmitters and sensory stimuli. The signalling and trafficking properties of GPCRs are often highly malleable depending on the cellular context. Such fine-tuning of GPCR function can be attributed in many cases to receptor-interacting proteins that are differentially expressed in distinct cell types. In some cases these GPCR-interacting partners directly mediate receptor signalling, whereas in other cases they act mainly as scaffolds to modulate G protein-mediated signalling. Furthermore, GPCR-interacting proteins can have a big impact on the regulation of GPCR trafficking, localization and/or pharmacological properties.