scholarly journals Developmentally Essential Protein Flightless I Is a Nuclear Receptor Coactivator with Actin Binding Activity

2004 ◽  
Vol 24 (5) ◽  
pp. 2103-2117 ◽  
Author(s):  
Young-Ho Lee ◽  
Hugh D. Campbell ◽  
Michael R. Stallcup
Keyword(s):  
Estrogen Receptor ◽  
Binding Site ◽  
Target Genes ◽  
Gene Promoter ◽  
Binding Activity ◽  
Actin Binding ◽  
Mouse Development ◽  
Methyltransferase Activity ◽  
Endogenous Estrogen ◽  
Coactivator Complex

ABSTRACT Hormone-activated nuclear receptors (NR) activate transcription by recruiting multiple coactivator complexes to the promoters of target genes. One important coactivator complex includes a p160 coactivator (e.g., GRIP1, SRC-1, or ACTR) that binds directly to activated NR, the histone acetyltransferase p300 or CBP, and the arginine-specific histone methyltransferase CARM1. We previously demonstrated that the coactivator function of CARM1 depends both on the methyltransferase activity and on additional unknown proteins that bind to CARM1. In this study a yeast two-hybrid screen for proteins that bind CARM1 identified the protein Flightless I (Fli-I), which has essential roles in Drosophila and mouse development. Fli-I bound to CARM1, GRIP1, and NRs and cooperated synergistically with CARM1 and GRIP1 to enhance NR function. Fli-I bound poorly to and did not cooperate with PRMT1, a CARM1-related protein arginine methyltransferase that also functions as an NR coactivator. The synergy between GRIP1, CARM1, and Fli-I required the methyltransferase activity of CARM1. The C-terminal AD1 (binding site for p300/CBP) and AD2 (binding site for CARM1) activation domains of GRIP1 contributed to the synergy but were less stringently required than the N-terminal region of GRIP1, which is the binding site for Fli-I. Endogenous Fli-I was recruited to the estrogen-regulated pS2 gene promoter of MCF-7 cells in response to the hormone, and reduction of endogenous Fli-I levels by small interfering RNA reduced hormone-stimulated gene expression by the endogenous estrogen receptor. A fragment of Fli-I that is related to the actin binding protein gelsolin enhanced estrogen receptor activity, and mutations that reduced actin binding also reduced the coactivator function of this Fli-I fragment. These data suggest that Fli-I may facilitate interaction of the p160 coactivator complex with other coactivators or coactivator complexes containing actin or actin-like proteins.

scholarly journals The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

2002 ◽  
Vol 13 (11) ◽  
pp. 3811-3821 ◽  
Author(s):  
Pauli J. Ojala ◽  
Ville O. Paavilainen ◽  
Maria K. Vartiainen ◽  
Roman Tuma ◽  
Alan G. Weeds ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Activity ◽  
Size Exclusion ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Monomer ◽  
Wild Type ◽  
Native Page ◽  
Exclusion Chromatography

Twinfilin is a ubiquitous and abundant actin monomer–binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K D = 0.05 μM) than ATP-G-actin (K D = 0.47 μM) under physiological ionic conditions and forms a relatively stable (k off = 1.8 s−1) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.

scholarly journals F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes.

1987 ◽  
Vol 105 (4) ◽  
pp. 1741-1751 ◽  
Author(s):  
L J Wuestehube ◽  
E J Luna
Keyword(s):  
Plasma Membrane ◽  
Binding Site ◽  
Dictyostelium Discoideum ◽  
Plasma Membranes ◽  
Membrane Binding ◽  
Binding Activity ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Cytoplasmic Surface ◽  
Actin Binding Site

F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin-membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge.

scholarly journals Isolation of Estrogen-Responsive Genes with a CpG Island Library

1998 ◽  
Vol 18 (1) ◽  
pp. 442-449 ◽  
Author(s):  
Toru Watanabe ◽  
Satoshi Inoue ◽  
Hisahiko Hiroi ◽  
Akira Orimo ◽  
Hiroyuki Kawashima ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Binding Site ◽  
Target Genes ◽  
Cpg Island ◽  
Cpg Islands ◽  
Human Cells ◽  
Receptor Protein ◽  
Upstream Region ◽  
Oxidase Subunit ◽  
Mcf 7

ABSTRACT In order to isolate novel estrogen-responsive genes, we utilized a CpG island library in which the regulatory regions of genes are enriched. CpG islands were screened for the ability to bind to a recombinant estrogen receptor protein with a genomic binding site (GBS) cloning method. Six CpG islands were selected, and they contained perfect, imperfect, and/or multiple half-palindromic estrogen-responsive elements (EREs). Northern blot analysis of various human cells showed that all these genomic fragments hybridized to specific mRNAs, suggesting that the genes associated with these EREs might be transcribed in human cells. Then cDNAs associated with two of them, EB1 and EB9, were isolated from libraries of human placenta and MCF-7 cells derived from a human breast cancer, respectively. Both transcripts were increased by estrogen in MCF-7 cells. The increase is inhibited by actinomycin D but not by cycloheximide, indicating that no protein synthesis is required for the up-regulation. The cDNA associated with EB1 encodes a 114-amino-acid protein similar to the cytochrome c oxidase subunit VIIa, named COX7RP (cytochromec oxidase subunit VII-related protein). The cDNA associated with EB9 is homologous only to an express sequence tag and was named EBAG9 (estrogen receptor-binding fragment-associated gene 9). The palindromic ERE of EB1 is located in an intron of COX7RP, and that of EB9 is in the 5′ upstream region of the cDNA. Both EREs had significant estrogen-dependent enhancer activities in a chloramphenicol acetyltransferase assay, when they were inserted into the 5′ upstream region of the chicken β-globin promoter. We therefore propose that the CpG-GBS method described here for isolation of the DNA binding site from the CpG island library would be useful for identification of novel target genes of certain transcription factors.

scholarly journals Rho GDP dissociation inhibitor α interacts with estrogen receptor α and influences estrogen responsiveness

2007 ◽  
Vol 39 (4) ◽  
pp. 249-259 ◽  
Author(s):  
Saad El Marzouk ◽  
Jennifer R Schultz-Norton ◽  
Varsha S Likhite ◽  
Ian X McLeod ◽  
John R Yates ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Target Genes ◽  
Estrogen Receptor Α ◽  
Negative Regulator ◽  
Guanosine Diphosphate ◽  
Estrogen Response ◽  
Endogenous Estrogen ◽  
Coregulatory Proteins ◽  
Gdp Dissociation Inhibitor ◽  
Mcf 7

AbstractEstrogen receptor α (ERα) is a ligand-activated transcription factor that regulates expression of estrogen-responsive genes. Upon binding of the ligand-occupied ERα to estrogen response elements (EREs) in DNA, the receptor interacts with a variety of coregulatory proteins to modulate transcription of target genes. We have isolated and identified a number of proteins associated with the DNA-bound ERα. One of these proteins, Rho guanosine diphosphate (GDP) dissociation inhibitor α (RhoGDIα), is a negative regulator of the Rho family of GTP-binding proteins. In this study, we demonstrate that endogenously expressed RhoGDIα is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, and that RhoGDIα binds directly to ERα, alters the ERα–ERE interaction, and influences the ability of ERα to regulate transcription of a heterologous estrogen-responsive reporter plasmid in transient transfection assays as well as endogenous, estrogen-responsive genes in MCF-7 cells. Our studies suggest that, in addition to the activity of RhoGDIα in the cytoplasm, it also influences ERα signaling in the nucleus.

Metabolite ligands of estrogen receptor-β reduce primate coronary hyperreactivity

2006 ◽  
Vol 290 (1) ◽  
pp. H295-H303 ◽  
Author(s):  
Rajesh G. Mishra ◽  
Frank Z. Stanczyk ◽  
Kenneth A. Burry ◽  
Suzanne Oparil ◽  
Benita S. Katzenellenbogen ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Binding Activity ◽  
Estrogen Receptor Β ◽  
Vascular Muscle ◽  
Vasoconstrictor Response ◽  
Endogenous Estrogen ◽  
17Β Estradiol ◽  
In Vitro Treatment

Previous reports showed that 17β-estradiol implants attenuate in vivo coronary hyperreactivity (CH), characterized by long-duration vasoconstrictions (in coronary angiographic experiments), in menopausal rhesus monkeys. Prolonged Ca2+ contraction signals that correspond with CH in coronary vascular muscle cells (VMC) to the same dual-constrictor stimulus, serotonin + the thromboxane analog U-46619, in estrogen-deprived VMC were suppressed by >72 h in 17β-estradiol. The purpose of this study was to test whether an endogenous estrogen metabolite with estrogen receptor-β (ER-β) binding activity, estriol (E3), suppresses in vivo and in vitro CH. E3 treatment in vivo for 4 wk significantly attenuated the angiographically evaluated vasoconstrictor response to intracoronary serotonin + U-46619 challenge. In vitro treatment of rhesus coronary VMC for >72 h with nanomolar E3 attenuated late Ca2+ signals. This reduction of late Ca2+ signals also appeared after >72 h of treatment with subnanomolar 5α-androstane-3β,17β-diol (3β-Adiol), an endogenous dihydrotestosterone metabolite with ER-β binding activity. R,R-tetrahydrochrysene, a selective ER-β antagonist, significantly blocked the E3- and 3β-Adiol-mediated attenuation of late Ca2+ signal increases. ER-β and thromboxane-prostanoid receptor (TPR) were coexpressed in coronary arteries and aorta. In vivo E3 treatment attenuated aortic TPR expression. Furthermore, in vitro treatment with E3 or 3β-Adiol downregulated TPR expression in VMC, which was blocked for both agonists by pretreatment with R,R-tetrahydrochrysene. E3- and 3β-Adiol-mediated reduction in persistent Ca2+ signals is associated with ER-β-mediated attenuation of TPR expression and may partly explain estrogen benefits in coronary vascular muscle.

Expression of Pit-1 and related proteins in diverse human pituitary adenomas

1993 ◽  
Vol 11 (3) ◽  
pp. 283-290 ◽  
Author(s):  
N Hoggard ◽  
K Callaghan ◽  
A Levy ◽  
J R E Davis
Keyword(s):  
Dna Binding ◽  
Pituitary Adenomas ◽  
Target Genes ◽  
Gene Promoter ◽  
Specific Protein ◽  
Binding Activity ◽  
Mobility Shift ◽  
Human Pituitary ◽  
Dna Binding Activity ◽  
Human Pituitary Adenomas

ABSTRACT Pit-1, a member of the POU family of homeodomain transcription factors, activates prolactin and GH gene expression but also has a role in pituitary cell differentiation and proliferation. Expression of Pit-1 may therefore be of central importance in the function and phenotype of human pituitary adenomas. We have found evidence that, in addition to Pit-1 mRNA, Pit-1-like immunoreactivity and DNA-binding activity are readily detectable in a series of human pituitary adenomas. Gel mobility shift assays using adenoma protein extracts with two Pit-1-binding sites from the human prolactin gene promoter demonstrated the formation of several DNA sequence-specific protein—DNA complexes; some of these could be accounted for by Oct-1-binding activity. Pit-1 activity was anticipated in prolactin- and GH-secreting adenomas, but was also detected in a proportion of endocrine-inactive (non-secreting) adenomas that did not express Pit-1 target genes. The data demonstrate the presence of Pit-1 in a range of pituitary adenomas. Different adenomas generated slightly differing patterns of DNA-binding activity, though Pit-1 mRNA and protein size appeared normal in all tumours so far examined.

scholarly journals GAC63, a GRIP1-Dependent Nuclear Receptor Coactivator

2005 ◽  
Vol 25 (14) ◽  
pp. 5965-5972 ◽  
Author(s):  
Yong-Heng Chen ◽  
Jeong Hoon Kim ◽  
Michael R. Stallcup
Keyword(s):  
Estrogen Receptor ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Gene Promoter ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Promoter Regions ◽  
Nuclear Receptor Coactivator ◽  
Binding Domains

ABSTRACT Nuclear receptors (NRs) regulate target gene transcription through the recruitment of multiple coactivator complexes to the promoter regions of target genes. One important coactivator complex includes a p160 coactivator (GRIP1, SRC-1, or ACTR) and its downstream coactivators (e.g., p300, CARM1, CoCoA, and Fli-I), which contribute to transcriptional activation by protein acetylation, protein methylation, and protein-protein interactions. In this study, we identified a novel NR coactivator, GAC63, which binds to the N-terminal region of p160 coactivators as well as the ligand binding domains of some NRs. GAC63 enhanced transcriptional activation by NRs in a hormone-dependent and GRIP1-dependent manner in transient transfection assays and cooperated synergistically and selectively with other NR coactivators, including GRIP1 and CARM1, to enhance estrogen receptor function. Endogenous GAC63 was recruited to the estrogen-responsive pS2 gene promoter of MCF-7 cells in response to the hormone. Reduction of the endogenous GAC63 level by small interfering RNA inhibited transcriptional activation by the hormone-activated estrogen receptor. Thus, GAC63 is a physiologically relevant part of the p160 coactivator signaling pathway that mediates transcriptional activation by NRs.

scholarly journals Study of Additional Interactions of Heterodimeric GW7604 Derivatives at the Coactivator Binding Site through Pharmacophore Modeling

2021 ◽  
Vol 9 (VI) ◽  
pp. 214-216
Author(s):  
Nilesh S. Kadu
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Binding Site ◽  
Clinical Stage ◽  
Endocrine Resistance ◽  
Pharmacophore Model ◽  
Structural Features ◽  
Pharmacophore Modeling ◽  
Binding Activity ◽  
Estrogen Receptor Modulators

Breast cancer is still the most common cancer in women worldwide, affecting one in eight women in high-income countries, and the incidence is further increasing. Endocrine therapy, including aromatase inhibitors or selective estrogen receptor modulators (SERMs)/selective estrogen receptor down-regulators (SERDs), consequently represents an indispensable treatment opportunity. Unfortunately, acquired endocrine resistance is an inevitable issue, which manifests after prolonged therapy. Consequently, developing a novel drug for the treatment of breast cancer is need of the hour. But it is an established fact that designing or repurposing a drug using ‘trial and error’ approach is a tricky, long, expensive and could be a failure in clinical stage. Hence, there is a need to employ alternative approaches like computer aided drug design (CADD) to overcome these shortcomings of conventional approach. Recently, CADD has gained a high popularity among drug designers and medicinal chemists due to several advances associated with it. Pharmacophore modeling is an efficient and useful approach to identify important patterns in a series of molecules for optimizations. Hence, in this analysis, an attempt is made to develop consensus pharmacophore model of heterodimeric GW7604 derivatives using alignment approach. The dataset consists of fourteen heterodimeric GW7604 derivatives exhibiting the binding activity in a transactivation assay ERα and ERβ to the coactivator binding site. The heterodimeric GW7604 derivatives possess good variation in substation pattern like the presence of different diaminoalkane spacer and CABS binder. The consensus pharmacophore model revealed the importance of structural features and their correlation with the biological activity.

scholarly journals Further characterization of the interaction between the cytoskeletal proteins talin and vinculin

2002 ◽  
Vol 362 (3) ◽  
pp. 761-768 ◽  
Author(s):  
Mark D. BASS ◽  
Bipin PATEL ◽  
Igor G. BARSUKOV ◽  
Ian J. FILLINGHAM ◽  
Robert MASON ◽  
...  
Keyword(s):  
Binding Site ◽  
Intramolecular Interaction ◽  
Binding Activity ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Yeast Two Hybrid ◽  
Temperature Dependent ◽  
Two Hybrid ◽  
Actin Binding Site

The cytoskeletal protein talin, which is thought to couple integrins to F-actin, contains three binding sites (VBS1—VBS3) for vinculin, a protein implicated in the negative regulation of cell motility and whose activity is modulated by an intramolecular interaction between the vinculin head (Vh) and vinculin tail (Vt) domains. In the present study we show that recombinant talin polypeptides containing the three VBSs (VBS1, residues 498–636; VBS2, residues 727–965; and VBS3, residues 1943–2157) each bind tightly to the same or overlapping sites within vinculin1–258. A short synthetic talin VBS3 peptide (residues 1944–1969) was sufficient to inhibit binding of a 125I-labelled talin VBS3 polypeptide to vinculin1–258, and NMR spectroscopy confirmed that this peptide forms a 1:1 complex in slow exchange with vinculin1–258. Binding of the 125I-labelled VBS3 polypeptide was markedly temperature dependent, but was not inhibited by 1M salt or 10% (v/v) 2-methyl-2-propanol. Attempts to further define the talin-binding site within vinculin1–258 using a gel-blot assay were unsuccessful, but near maximal talin-binding activity was retained by a construct spanning vinculin residues 1–131 in a yeast two-hybrid assay. Interestingly, the talin VBS3 polypeptide was a potent inhibitor of the Vh—Vt interaction, and the VBS3 synthetic peptide was able to expose the actin-binding site in intact vinculin, which is otherwise masked by the Vh—Vt interaction. The results suggest that under certain conditions, talin may be an effective activator of vinculin.

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