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Ligand Binding Domain (ligand + binding_domain)
Selected AbstractsMolecular and functional characterization of novel CRFR1 isoforms from the skinFEBS JOURNAL, Issue 13 2004Alexander Pisarchik In our continued studies on corticotropin releasing factor receptor (CRFR1) signaling in the skin, we tested functional activity of CRFR1,, e, f, g and h isoforms after transfection to COS cells. Both membrane-bound and soluble variants are translated in vivo into final protein products that undergo further post-translational modifications. CRFR1, was the only isoform coupled directly to adenylate cyclase with the exception of an artificial isoform (CRFR1h2) with the insertion of 37 amino acids between the ligand binding domain and the first extracellular loop that was capable of producing detectable levels of cyclic AMP (cAMP). Soluble isoforms could modulate cell response with CRFR1e attenuating and CRFR1h amplifying CRFR1,-coupled cAMP production stimulated by urocortin. Testing with plasmids containing the luciferase reporter gene, and inducible cis -elements (CRE, CaRE, SRE, AP1 or NF-,B) demonstrated that only CRFR1, was involved directly in the transcriptional regulation, while CRFR1g inhibited CRE activity. Significantly higher reporter gene expression by CRF was observed than that mediated by 4,-phorbol 12-myristate 13-acetate and forskolin alone, being compatible with the concomitant treatment by phorbol 12-myristate 13-acetate and forskolin. This suggests that both protein kinase A and C can be involved in CRF-dependent signal transduction. [source] Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracleFEBS JOURNAL, Issue 13 2002Markus Lezzi The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner. This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development. [source] Molecular cloning and expression of Tenebrio molitor ultraspiracle during metamorphosis and in vivo induction of its phosphorylation by 20-hydroxyecdysoneINSECT MOLECULAR BIOLOGY, Issue 3 2000M. Nicolaļ Abstract Using a RT-PCR approach, the Tenebrio molitor homologue of Drosophila Ultraspiracle (TmUSP) was characterized. Its DNA binding domain shows a degree of identity with those of the other insect USPs. However, the ligand binding domain is closer to those of retinoid X receptors. Using an antibody raised against DmUSP, Western blot analysis of proteins from epidermis and other tissues revealed five immunoreactive bands, corresponding to different phosphorylated forms of a unique polypeptide, as shown by ,-phosphatase treatment. The nuclear form of TmUSP seems unphosphorylated. An in vivo 20-hydroxyecdysone treatment increases considerably and rapidly the phosphorylated forms of TmUSP. This post-translational modification may play a role in the 20-hydroxyecdysone response. [source] Detection and localization of an estrogen receptor beta splice variant protein (ER,2) in the adult female rat forebrain and midbrain regionsTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 3 2007Wilson C.J. Chung Abstract Estrogens regulate neural processes such as neuronal development, reproductive behavior, and hormone secretion, and signal through estrogen receptor (ER) , and ER, (here called ER,1). Recent studies have found variations in ER, and ER,1 mRNA splicing in rodents and humans. Functional reporter gene assays suggest that these splicing variations alter ER-mediated transcriptional regulation. Estrogen receptor beta 2 (ER,2), an ER,1 splice variant containing an 18 amino acid (AA) insert in the ligand binding domain, binds estradiol with ,10-fold lower affinity than ER,1, suggesting that it may serve as a low-affinity ER. Moreover, ER,2 reportedly acts in a dominant-negative fashion when heterodimerized with ER,1 or ER,. To explore the function of ER,2 in brain, an antiserum (Two,ER.1) targeting the 18 AA insert was developed and characterized. Western blot analysis and transient expression of ER,2 in cell lines demonstrated that Two,ER.1 recognizes ER,2. In the adult female rat brain, ER,2 immunoreactivity is localized in the cell nucleus and is expressed with a distribution similar to that of ER,1 mRNA. ER,2 immunoreactive cell numbers were high in, for example, piriform cortex, paraventricular nucleus, supraoptic nucleus, arcuate nucleus, and hippocampal CA regions, whereas it was low in the dentate gyrus. Moreover, ER,2 is coexpressed in gonadotropin-releasing hormone and oxytocin neurons. These studies demonstrate ER, splice variant proteins in brain and support the hypothesis that ER signaling diversity depends not only on ligand or coregulatory proteins, but also on regional and phenotypic selectivity of ER splice variant proteins. J. Comp. Neurol. 505:249,267, 2007. © 2007 Wiley-Liss, Inc. [source] ERAP75 functions as a coactivator to enhance estrogen receptor , transactivation in prostate stromal cells,THE PROSTATE, Issue 12 2008Ming Chen Abstract BACKGROUND Estrogen receptor , (ER,) has been reported to be expressed and function in the prostate stromal cells, and numerous evidences indicated that the stromal ER, signal pathway plays critical roles in prostate development and cancer. ER, requires distinct coregulators for efficient transcriptional regulation. The goal of this study is to examine physical and functional interaction between ER, and ERAP75 in the context of prostate stromal cells. METHOD Yeast two-hybrid assays were used to screen novel ER, interaction proteins. The interaction between ER, and ERAP75 was confirmed by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation methods. The interaction motif was examined by site-directed mutagenesis. The effect of ERAP75 on ER, transactivation and the expression of ER, target genes were determined by luciferase assay and real-time PCR, respectively. RESULT ER, can interact with the C terminus of ERAP75 via its ligand binding domain both in vivo and in vitro. The conserved LXXLL motif within the C terminus of ERAP75 is required for the interaction between ER, and ERAP75. ERAP75 can enhance ER, transactivation in a dose-dependent manner and up-regulate the expression of the endogenous ER, target gene, stromal-derived factor-1 (SDF-1), in the prostate stromal cells. CONCLUSION ERAP75 functions as a novel coactivator that can modulate ER, function in the prostate stromal cells. The understanding of the mechanism of ER, transactivation in prostate stromal cells could possibly help in the development of new strategies to control or treat prostate cancer by targeting its transactivation protein complex. Prostate 68:1273,1282, 2008. © 2008 Wiley-Liss, Inc. [source] Structure of the Glucocorticoid Receptor, a Flexible Protein That Can Adapt to Different LigandsCHEMMEDCHEM, Issue 5 2010Adriana Crystal structures of the glucocorticoid receptor (GR) ligand binding domain in complex with various agonists and antagonists give us an insight on how ligands are recognized by the receptor and how their structure can affect the behavior of the GR. Interestingly, these structural data show how the GR can adapt its binding pocket to accommodate molecules that differ substantially from the natural ligands without loss of function. [source] Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracleFEBS JOURNAL, Issue 13 2002Markus Lezzi The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner. This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development. [source] | |||||||||||||