scholarly journals Human Glucocorticoid Receptor β Binds RU-486 and Is TranscriptionallyActive

2007 ◽  
Vol 27 (6) ◽  
pp. 2266-2282 ◽  
Author(s):  
Laura J. Lewis-Tuffin ◽  
Christine M. Jewell ◽  
Rachelle J. Bienstock ◽  
Jennifer B. Collins ◽  
John A. Cidlowski
Keyword(s):  
Confocal Microscopy ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Orphan Receptor ◽  
Computational Docking ◽  
Ru 486

ABSTRACT Human glucocorticoid receptor (hGR) is expressed as two alternately spliced C-terminal isoforms, α and β. In contrast to the canonical hGRα, hGRβ is a nucleus-localized orphan receptor thought not to bind ligand and not to affect gene transcription other than by acting as a dominant negative to hGRα. Here we used confocal microscopy to examine the cellular localization of transiently expressed fluorescent protein-tagged hGRβ in COS-1 and U-2 OS cells. Surprisingly, yellow fluorescent protein (YFP)-hGRβ was predominantly located in the cytoplasm and translocated to the nucleus following application of the glucocorticoid antagonist RU-486. This effect of RU-486 was confirmed with transiently expressed wild-type hGRβ. Confocal microscopy of coexpressed YFP-hGRβ and cyan fluorescent protein-hGRα in COS-1 cells indicated that the receptors move into the nucleus independently. Using a ligand binding assay, we confirmed that hGRβ bound RU-486 but not the hGRα ligand dexamethasone. Examination of the cellular localization of YFP-hGRβ in response to a series of 57 related compounds indicated that RU-486 is thus far the only identified ligand that interacts with hGRβ. The selective interaction of RU-486 with hGRβ was also supported by molecular modeling and computational docking studies. Interestingly, microarray analysis indicates that hGRβ, expressed in the absence of hGRα, can regulate gene expression and furthermore that occupation of hGRβ with the antagonist RU-486 diminishes that capacity despite the lack of helix 12 in the ligand binding domain.

scholarly journals Probing Dominant Negative Behavior of Glucocorticoid Receptor β through a Hybrid Structural and Biochemical Approach

2018 ◽  
Vol 38 (8) ◽  
pp. e00453-17 ◽  
Author(s):  
Jungki Min ◽  
Lalith Perera ◽  
Juno M. Krahn ◽  
Christine M. Jewell ◽  
Andrea F. Moon ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Binding Pocket ◽  
Dominant Negative ◽  
Ru 486 ◽  
Dominant Negative Inhibitor ◽  
Helix 12 ◽  
Binding Energy Analysis ◽  
Biochemical Approach ◽  
Biochemical Analyses

ABSTRACT Glucocorticoid receptor β (GRβ) is associated with glucocorticoid resistance via dominant negative regulation of GRα. To better understand how GRβ functions as a dominant negative inhibitor of GRα at a molecular level, we determined the crystal structure of the ligand binding domain of GRβ complexed with the antagonist RU-486. The structure reveals that GRβ binds RU-486 in the same ligand binding pocket as GRα, and the unique C-terminal amino acids of GRβ are mostly disordered. Binding energy analysis suggests that these C-terminal residues of GRβ do not contribute to RU-486 binding. Intriguingly, the GRβ/RU-486 complex binds corepressor peptide with affinity similar to that of a GRα/RU-486 complex, despite the lack of helix 12. Our biophysical and biochemical analyses reveal that in the presence of RU-486, GRβ is found in a conformation that favors corepressor binding, potentially antagonizing GRα function. This study thus presents an unexpected molecular mechanism by which GRβ could repress transcription.

Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm

Development ◽  
2000 ◽  
Vol 127 (4) ◽  
pp. 791-800 ◽  
Author(s):  
M. Kishi ◽  
K. Mizuseki ◽  
N. Sasai ◽  
H. Yamazaki ◽  
K. Shiota ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Cell Fate ◽  
Neural Differentiation ◽  
Dominant Negative ◽  
Gastrula Stage ◽  
Blastula Stage ◽  
Sensory Epithelia ◽  
Neural Tissues

From early stages of development, Sox2-class transcription factors (Sox1, Sox2 and Sox3) are expressed in neural tissues and sensory epithelia. In this report, we show that Sox2 function is required for neural differentiation of early Xenopus ectoderm. Microinjection of dominant-negative forms of Sox2 (dnSox2) mRNA inhibits neural differentiation of animal caps caused by attenuation of BMP signals. Expression of dnSox2 in developing embryos suppresses expression of N-CAM and regional neural markers. We have analyzed temporal requirement of Sox2-mediated signaling by using an inducible dnSox2 construct fused to the ligand-binding domain of the glucocorticoid receptor. Attenuation of Sox2 function both from the late blastula stage and from the late gastrula stage onwards causes an inhibition of neural differentiation in animal caps and in whole embryos. Additionally, dnSox2-injected cells that fail to differentiate into neural tissues are not able to adopt epidermal cell fate. These data suggest that Sox2-class genes are essential for early neuroectoderm cells to consolidate their neural identity during secondary steps of neural differentiation.

scholarly journals Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay

2019 ◽  
Vol 20 (16) ◽  
pp. 3859 ◽  
Author(s):  
Michael Winkler ◽  
Florian Wrensch ◽  
Pascale Bosch ◽  
Maike Knoth ◽  
Michael Schindler ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Protein Interactions ◽  
Viral Entry ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions

The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

scholarly journals The NHERF1 PDZ1 domain and IRBIT interact and mediate the activation of Na+/H+ exchanger 3 by ANG II

2016 ◽  
Vol 311 (2) ◽  
pp. F343-F351 ◽  
Author(s):  
Peijian He ◽  
Luqing Zhao ◽  
Yi Ran No ◽  
Serhan Karvar ◽  
C. Chris Yun
Keyword(s):  
Proximal Tubule ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Yellow Fluorescent Protein ◽  
Protein S ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Ang Ii ◽  
Renal Brush Border Membrane ◽  
Inositol 1,4,5 Trisphosphate

Na+/H+ exchanger (NHE)3, a major Na+ transporter in the luminal membrane of the proximal tubule, is subject to ANG II regulation in renal Na+/fluid absorption and blood pressure control. We have previously shown that inositol 1,4,5-trisphosphate receptor-binding protein released with inositol 1,4,5-trisphosphate (IRBIT) mediates ANG II-induced exocytosis of NHE3 in cultured proximal tubule epithelial cells. In searching for scaffold protein(s) that coordinates with IRBIT in NHE3 trafficking, we found that NHE regulatory factor (NHERF)1, NHE3, and IRBIT proteins were coexpressed in the same macrocomplexes and that loss of ANG II type 1 receptors decreased their expression in the renal brush-border membrane. We found that NHERF1 was required for ANG II-mediated forward trafficking and activation of NHE3 in cultured cells. ANG II induced a concomitant increase of NHERF1 interactions with NHE3 and IRBIT, which were abolished when the NHERF1 PDZ1 domain was removed. Overexpression of a yellow fluorescent protein-NHERF1 construct that lacks PDZ1, but not PDZ2, failed to exaggerate the ANG II-dependent increase of NHE3 expression in the apical membrane. Moreover, exogenous expression of PDZ1 exerted a dominant negative effect on NHE3 activation by ANG II. We further demonstrated that IRBIT was indispensable for the ANG II-provoked increase in NHERF1-NHE3 interactions and that phosphorylation of IRBIT at Ser68 was necessary for the assembly of the NHEF1-IRBIT-NHE3 complex. Taken together, our findings suggest that NHERF1 mediates ANG II-induced activation of renal NHE3, which requires coordination between IRBIT and the NHERF1 PDZ1 domain in binding and transporting NHE3.

scholarly journals Actin Depolymerization Disrupts Tight Junctions via Caveolae-mediated Endocytosis

2005 ◽  
Vol 16 (9) ◽  
pp. 3919-3936 ◽  
Author(s):  
Le Shen ◽  
Jerrold R. Turner
Keyword(s):  
Barrier Function ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Yellow Fluorescent Protein ◽  
Membrane Traffic ◽  
Time Lapse ◽  
Dominant Negative ◽  
Enhanced Yellow Fluorescent Protein ◽  
Caveolin 1 ◽  
Actin Depolymerization

The tight junction (TJ) determines epithelial barrier function. Actin depolymerization disrupts TJ structure and barrier function, but the mechanisms of this effect remain poorly understood. The goal of this study was to define these mechanisms. Madin-Darby canine kidney (MDCK) cells expressing enhanced green fluorescent protein-, enhanced yellow fluorescent protein-, or monomeric red fluorescent protein 1-fusion proteins of β-actin, occludin, claudin-1, ZO-1, clathrin light chain A1, and caveolin-1 were imaged by time-lapse multidimensional fluorescence microscopy with simultaneous measurement of transepithelial electrical resistance (TER). Actin depolymerization was induced with latrunculin A (LatA). Within minutes of LatA addition TER began to fall. This coincided with occludin redistribution and internalization. In contrast, ZO-1 and claudin-1 redistribution occurred well after maximal TER loss. Occludin internalization and TER loss, but not actin depolymerization, were blocked at 14°C, suggesting that membrane traffic is required for both events. Inhibition of membrane traffic with 0.4 M sucrose also blocked occludin internalization and TER loss. Internalized occludin colocalized with caveolin-1 and dynamin II, but not with clathrin, and internalization was blocked by dominant negative dynamin II (K44A), but not by Eps15Δ95-295 expression. Inhibition of caveolae-mediated endocytosis by cholesterol extraction prevented both LatA-induced TER loss and occludin internalization. Thus, LatA-induced actin depolymerization causes TJ structural and functional disruption by mechanisms that include caveolae-mediated endocytosis of TJ components.

scholarly journals A Novel, C-Terminal Dominant Negative Mutation of the GR Causes Familial Glucocorticoid Resistance through Abnormal Interactions with p160 Steroid Receptor Coactivators

2002 ◽  
Vol 87 (6) ◽  
pp. 2658-2667 ◽  
Author(s):  
Alessandra Vottero ◽  
Tomoshige Kino ◽  
Herve Combe ◽  
Pierre Lecomte ◽  
George P. Chrousos
Keyword(s):  
Ligand Binding ◽  
Transcriptional Activity ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Glucocorticoid Resistance ◽  
Base Substitution ◽  
Wild Type ◽  
Nuclear Speckles ◽  
Dominant Negative Mutation ◽  
Type Receptor

Primary cortisol resistance is a rare, inherited or sporadic form of generalized end-organ insensitivity to glucocorticoids. Here, we report a kindred in which affected members had a heterozygous T to G base substitution at nucleotide 2373 of exon 9α of the GR gene, causing substitution of Ile by Met at position 747. This mutation was located close to helix 12, at the C terminus of the ligand-binding domain, which has a pivotal role in the formation of activation function (AF)-2, a subdomain that interacts with p160 coactivators. The affinity of the mutant GR for dexamethasone was decreased by about 2-fold, and its transcriptional activity on the glucocorticoid-responsive mouse mammary tumor virus promoter was compromised by 20- to 30-fold. In addition, the mutant GR functioned as a dominant negative inhibitor of wild-type receptor-induced transactivation. The mutant GR through its intact AF-1 domain bound to a p160 coactivator, but failed to do so through its AF-2 domain. Overexpression of a p160 coactivator restored the transcriptional activity and reversed the negative transdominant activity of the mutant GR. Interestingly, green fluorescent protein (GFP)-fused GRαI747M had a slight delay in its translocation from the cytoplasm into the nucleus and formed coarser nuclear speckles than GFP-fused wild-type GRα. Similarly, a GFP-fused p160 coactivator had a distinctly different distribution in the nucleus in the presence of mutant vs. wild-type receptor, presenting also as coarser speckling. We conclude that the mutation at amino acid 747 of the GR causes familial, autosomal dominant glucocorticoid resistance by decreasing ligand binding affinity and transcriptional activity, and by exerting a negative transdominant effect on the wild-type receptor. The mutant receptor has an ineffective AF-2 domain, which leads to an abnormal interaction with p160 coactivators and a distinct nuclear distribution of both.

scholarly journals NBCe1A dimer assemble visualized by bimolecular fluorescence complementation

2014 ◽  
Vol 306 (6) ◽  
pp. F672-F680 ◽  
Author(s):  
Min-Hwang Chang ◽  
An-Ping Chen ◽  
Michael F. Romero
Keyword(s):  
Fluorescent Protein ◽  
Functional Unit ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Bimolecular Fluorescence Complementation ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Enhanced Yellow Fluorescent Protein ◽  
Type Function ◽  
Fluorescence Complementation

Mutations in the electrogenic Na+/HCO3−cotransporter (NBCe1) that cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts in patients are recessive. Parents and siblings of these affected individuals seem asymptomatic although their tissues should make some mutant NBCe1 protein. Biochemical studies with AE1 and NBCe1 indicate that both, and probably all, Slc4 members form dimers. However, the physiologic implications of dimerization have not yet been fully explored. Here, human NBCe1A dimerization is demonstrated by biomolecular fluorescence complementation (BiFC). An enhanced yellow fluorescent protein (EYFP) fragment (1–158, EYFPN) or (159–238, EYFPC) was fused to the NH2or COOH terminus of NBCe1A and mix-and-matched expressed in Xenopus oocyte. The EYFP fluorescent signal was observed only when both EYFP fragments are fused to the NH2terminus of NBCe1A (EYFPN-N-NBCe1A w/ EYFPC-N-NBCe1A), and the electrophysiology data demonstrated this EYFP-NBCe1A coexpressed pair have wild-type transport function. These data suggest NBCe1A forms dimers and that NH2termini from the two monomers are in close proximity, likely pair up, to form a functional unit. To explore the physiologic significance of NBCe1 dimerization, we chose two severe NBCe1 mutations (6.6 and 20% wild-type function individually): S427L (naturally occurring) and E91R (for NH2-terminal structure studies). When we coexpressed S427L and E91R, we measured 50% wild-type function, which can only occur if the S427L-E91R heterodimer is the functional unit. We hypothesize that the dominant negative effect of heterozygous NBCe1 carrier should be obvious if the mutated residues are structurally crucial to the dimer formation. The S427L-E91R heterodimer complex allows the monomers to structurally complement each other resulting in a dimer with wild-type like function.

scholarly journals Epidermolysis Bullosa Simplex-Type Mutations Alter the Dynamics of the Keratin Cytoskeleton and Reveal a Contribution of Actin to the Transport of Keratin Subunits

2004 ◽  
Vol 15 (3) ◽  
pp. 990-1002 ◽  
Author(s):  
Nicola Susann Werner ◽  
Reinhard Windoffer ◽  
Pavel Strnad ◽  
Christine Grund ◽  
Rudolf Eberhard Leube ◽  
...  
Keyword(s):  
Epidermolysis Bullosa ◽  
Fluorescent Protein ◽  
Dynamic Equilibrium ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Cell Periphery ◽  
Epidermolysis Bullosa Simplex ◽  
Enhanced Yellow Fluorescent Protein ◽  
Dominant Negative Mutation

Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.

Neurotensin receptor type 1: Escherichia coli expression, purification, characterization and biophysical study

2007 ◽  
Vol 35 (4) ◽  
pp. 760-763 ◽  
Author(s):  
P.J. Harding ◽  
H. Attrill ◽  
S. Ross ◽  
J.R. Koeppe ◽  
A.N. Kapanidis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Single Molecule ◽  
Fluorescent Protein ◽  
Radioligand Binding ◽  
Yellow Fluorescent Protein ◽  
Active Form ◽  
X Ray Crystallography ◽  
Biophysical Study ◽  
Escherichia Coli Expression

NT (neurotensin) is an endogenous tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. One receptor for NT, NTS1, belongs to the GPCR (G-protein-coupled receptor) superfamily, has seven putative transmembrane domains, and is being studied by a range of single-molecule, functional and structural approaches. To enable biophysical characterization, sufficient quantities of the receptor need to be expressed and purified in an active form. To this end, rat NTS1 has been expressed in Escherichia coli in an active ligand-binding form at the cell membrane and purified in sufficient amounts for structural biology studies either with or without fluorescent protein [YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein)] fusions. Ligand binding has been demonstrated in a novel SPR (surface plasmon resonance) approach, as well as by conventional radioligand binding measurements. These improvements in production of NTS1 now open up the possibility of direct structural studies, such as solid-state NMR to interrogate the NT-binding site, EM (electron microscopy), and X-ray crystallography and NMR.

scholarly journals Probing the domain structure of FtsZ by random truncation and insertion of GFP

Microbiology ◽  
2005 ◽  
Vol 151 (12) ◽  
pp. 4033-4043 ◽  
Author(s):  
Masaki Osawa ◽  
Harold P. Erickson
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Instability ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Wild Type ◽  
Negative Effects ◽  
Terminal Domain ◽  
Z Ring ◽  
Green Fluorescent

Random transposon-mediated mutagenesis has been used to create truncations and insertions of green fluorescent protein (GFP), and Venus-yellow fluorescent protein (YFP), in Escherichia coli FtsZ. Sixteen unique insertions were obtained, and one of them, in the poorly conserved C-terminal spacer, was functional for cell division with the Venus-YFP insert. The insertion of enhanced GFP (eGFP) at this same site was not functional; Venus-YFP was found to be superior to eGFP in other respects too. Testing the constructs for dominant negative effects led to the following general conclusion. The N-terminal domain, aa 1–195, is an independently folding domain that can poison Z-ring function when expressed without a functional C-terminal domain. The effects were weak, requiring expression of the mutant at 3–5 times the level of wild-type FtsZ. The C-terminal domain, aa 195–383, was also independently folding, but had no activity in vivo. The differential activity of the N- and C-terminal domains suggests that FtsZ protofilament assembly is directional, with subunits adding primarily at the bottom of the protofilament. Directional assembly could occur by either a treadmilling or a dynamic instability mechanism.

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