RESUMEN
A novel, retinoic acid-induced gene, GRP1-associated scaffold protein (GRASP), was isolated from P19 embryonal carcinoma cells using a subtractive screening strategy. GRASP was found to be highly expressed in brain and exhibited lower levels of expression in lung, heart, embryo, kidney, and ovary. The predicted amino acid sequence of GRASP is characterized by several putative protein-protein interaction motifs, suggesting that GRASP may be a component of a larger protein complex in the cell. Although GRASP does not harbor a predicted membrane spanning domain(s), the protein was observed to be associated with the plasma membrane of transiently transfected mammalian cells. Yeast two-hybrid screening revealed that GRASP interacted strongly with the General Receptor for Phosphoinositides 1 (GRP1), a brefeldin A-insensitive guanine nucleotide exchange factor for the ADP-ribosylation factor family of proteins. GRASP. GRP1 interactions were also demonstrated in vitro and in mammalian cells in which GRASP was shown to enhance GRP1 association with the plasma membrane. Furthermore, GRASP colocalized with endogenous ADP-ribosylation factors at the plasma membrane in transfected cells, suggesting that GRASP may modulate signaling by this family of small GTPases.
Asunto(s)
Proteínas Portadoras/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Tretinoina/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Extractos Celulares , Clonación Molecular , ADN Complementario , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de la Membrana , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Two novel and related C(2)H(2) zinc finger proteins that are highly expressed in the brain, CTIP1 and CTIP2 (COUP TF-interacting proteins 1 and 2, respectively), were isolated and shown to interact with all members of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of orphan nuclear receptors. The interaction of CTIP1 with ARP1 was studied in detail, and CTIP1 was found to harbor two independent ARP1 interaction domains, ID1 and ID2, whereas the putative AF-2 of ARP1 was required for interaction with CTIP1. CTIP1, which exhibited a punctate staining pattern within the nucleus of transfected cells, recruited cotransfected ARP1 to these foci and potentiated ARP1-mediated transcriptional repression of a reporter construct. However, transcriptional repression mediated by ARP1 acting through CTIP1 did not appear to involve recruitment of a trichostatin A-sensitive histone deacetylase(s) to the template, suggesting that this repression pathway may be distinct from that utilized by several other nuclear receptors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Ovalbúmina/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides , Factores de Transcripción/metabolismo , Transcripción Genética , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Factor de Transcripción COUP I , Factor de Transcripción COUP II , Factores de Transcripción COUP , Núcleo Celular/metabolismo , Pollos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Embrión de Mamíferos , Embrión no Mamífero , Regulación de la Expresión Génica , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/farmacología , Masculino , Ratones , Datos de Secuencia Molecular , Mutagénesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Eliminación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/genética , TransfecciónRESUMEN
Nuclear receptor corepressor (NCoR) was demonstrated to interact strongly with peroxisome proliferator-activated receptor alpha (PPARalpha), and PPARalpha ligands suppressed this interaction. In contrast to the interaction of PPARalpha with the coactivator protein, p300, association of the receptor with NCoR did not require any part of the PPARalpha ligand binding domain. NCoR was found to suppress PPARalpha-dependent transcriptional activation in the context of a PPARalpha.retinoid X receptor alpha (RXRalpha) heterodimeric complex bound to a peroxisome proliferator-responsive element in human embryonic kidney 293 cells. This repression was reversed agonists of either receptor demonstrating a functional interaction between NCoR and PPARalpha.RXRalpha heterodimeric complexes in mammalian cells. NCoR appears to influence PPARalpha signaling pathways and, therefore, may modulate tissue responsiveness to peroxisome proliferators.
Asunto(s)
Microcuerpos/metabolismo , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Clonación Molecular , Dimerización , Humanos , Ligandos , Datos de Secuencia Molecular , Mutación/genética , Co-Represor 1 de Receptor Nuclear , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Proteínas Represoras/genética , Receptores X Retinoide , Transducción de Señal/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Transfección/genética , Levaduras/genética , Receptor de Ácido Retinoico gammaRESUMEN
Members of the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) subfamily of orphan nuclear receptors, which minimally includes COUP-TFI and ARP1, are highly expressed in brain and are generally considered to be constitutive repressors of transcription. We have used a yeast two-hybrid system to isolate proteins expressed in brain that interact with ARP1. One of the proteins isolated in this screen was Ear2, another orphan receptor that has been suggested to be a member of the COUP-TF subfamily. Here we demonstrate that ARP1 and Ear2 form heterodimers in solution and on directly repeated response elements with high efficiency and a specificity differing from that of homodimeric complexes composed of either receptor. ARP1 and Ear2 were observed to interact in mammalian cells, and the tissue distribution of Ear2 transcripts was found to overlap precisely with the expression pattern of ARP1 in several mouse tissues and embryonal carcinoma cell lines. Heterodimeric interactions between ARP1 and Ear2 may define a distinct pathway of orphan receptor signaling.
Asunto(s)
Proteínas Aviares , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides , Factores de Transcripción/metabolismo , Animales , Factor de Transcripción COUP I , Factor de Transcripción COUP II , Factores de Transcripción COUP , Línea Celular , Pollos , Dimerización , Humanos , Ovalbúmina/genética , Ovalbúmina/metabolismo , Unión Proteica , Proteínas RepresorasRESUMEN
The integrator protein, p300, was demonstrated to interact with mouse peroxisome proliferator-activated receptor alpha in a ligand-enhanced manner. The PPARalpha-interacting domain of p300 was mapped to amino acids 39-117 which interacted strongly with PPARalpha but did not interact with retinoic acid receptor-gamma or retinoid X receptor-alpha. Amino acids within the carboxyl terminus of PPARalpha as well as residues within the hinge region were required for ligand-dependent interaction with p300. p300 enhanced the transcriptional activation properties of PPARalpha and, therefore, can be considered a bona fide coactivator for this nuclear receptor. These observations extend the group of p300-interacting proteins to include mPPARalpha and further characterize the molecular mechanisms of PPARalpha-mediated transcriptional regulation.