Filter-binding assay procedure for thyroid hormone receptors

Inoue, Akira; Yamakawa, J; Yukioka, Munehiko; Morisawa, Seiji

doi:10.1016/0003-2697(83)90280-4

Cited by 82 publications

(35 citation statements)

References 17 publications

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“…Future studies should be performed to compare strategies with the wild-type and mutant systems. To date, several in vitro assays have been developed for screening ER ligands by using either purified ER␣ protein or ER isolated from cell lysates (18)(19)(20)(21). Limited fluorescence-based assays (22) have been developed to measure receptor conformational changes (23) and recruitment of coactivator peptides (22,24,25) in the full-length hER␣ within cell culture (26).…”

Section: Discussionmentioning

confidence: 99%

An intramolecular folding sensor for imaging estrogen receptor–ligand interactions

Paulmurugan

Gambhir

2006

Proc. Natl. Acad. Sci. U.S.A.

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Strategies for high-throughput analysis of interactions between various hormones and drugs with the estrogen receptor (ER) are crucial for accelerating the understanding of ER biology and pharmacology. Through careful analyses of the crystal structures of the human ER (hER) ligand-binding domain (hER-LBD) in complex with different ligands, we hypothesized that the hER-LBD intramolecular folding pattern could be used to distinguish ER agonists from selective ER modulators and pure antiestrogens. We therefore constructed and validated intramolecular folding sensors encoding various hER-LBD fusion proteins that could lead to split Renilla͞firefly luciferase reporter complementation in the presence of the appropriate ligands. A mutant hER-LBD with low affinity for circulating estradiol was also identified for imaging in living subjects. Cells stably expressing the intramolecular folding sensors expressing wild-type and mutant hER-LBD were used for imaging ligand-induced intramolecular folding in living mice. This is the first hER-LBD intramolecular folding sensor suited for high-throughput quantitative analysis of interactions between hER with hormones and drugs using cell lysates, intact cells, and molecular imaging of small living subjects. The strategies developed can also be extended to study and image other important protein intramolecular folding systems.complementation ͉ optical imaging ͉ split reporters E strogens are responsible for the growth, development, and maintenance of the reproductive, skeletal, neuronal, and immune systems as well as and other systems. The physiological effects of these hormones are mediated by the estrogen receptor (ER), which is a ligand-inducible nuclear transcription factor (1). In the classical pathway of steroid hormone action, 17␤-estradiol (E2), hormones, and a variety of other estrogens bind to the ligandbinding domain (LBD) of ER and lead to its dimerization and subsequent binding to a specific regulatory sequence in the promoters of ER target genes known as the estrogen response elements (2, 3), which then trigger activation or repression of many downstream target genes (4). The deficiency or excess of estrogens can lead to various pathological conditions including osteoporosis and breast carcinomas (5), making ER a major cellular therapeutic target.Elegant crystallographic studies with ER-LBD have shown that conformation of helix 12 (H12) is critical in responses observed with various ER ligands (4, 6, 7). The conformation of H12 behaves as a ''molecular switch'' that either prevents or enhances ER from binding to an array of coactivator proteins, which then activates transcription of many downstream estrogen-regulated genes responsible for cell growth. Given the critical role of H12 in ER signaling, we reasoned that it may be feasible to develop an intramolecular ER folding sensor with specific split reporter complementation patterns to study ligand pharmacology based directly on the conformational changes of H12 in response to different ligands (Fig. 1).We used a spli...

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Section: Discussionmentioning

confidence: 99%

An intramolecular folding sensor for imaging estrogen receptor–ligand interactions

Paulmurugan

Gambhir

2006

Proc. Natl. Acad. Sci. U.S.A.

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“…T3 binding assays were done essentially as described before (19). Cell-free translated T3R␣ and/or RXR␣ was incubated on ice for 15 min.…”

Section: Methodsmentioning

confidence: 99%

A shift in the Ligand Responsiveness of Thyroid Hormone Receptor α Induced by Heterodimerization with Retinoid X Receptor α

Claret

Antakly

Karin

et al. 1996

Molecular and Cellular Biology

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Thyroid hormone (T3) receptors (T3Rs) are ligand-modulated transcription factors that bind to thyroid hormone response elements (T3REs) and mediate either positive or negative transcriptional regulation of target genes. In addition, in response to ligand binding, T3Rs can interfere with AP-1 activity and thereby inhibit transcription of AP-1-responsive genes. T3Rs were recently shown to form heterodimers with retinoid X receptors (RXRs), leading to increased binding to T3REs in vitro and potentiation of transcriptional responses in vivo. Here we demonstrate that T3R␣ forms stable heterodimers with RXR␣ in living cells. Most important, we describe a new role for RXR␣ in modulating ligand-dependent T3R␣ activity: heterodimerization with RXR␣ greatly increases transcriptional interference with AP-1 activity, augments T3-dependent transcriptional activation, and potentiates the reversal of ligand-independent activation by T3R␣. In all three cases, the responses occur at substantially lower T3 concentrations when elicited by T3R␣ plus RXR␣ than by T3R␣ alone. In vitro, the binding of T3 decreases the DNA-binding activity of T3R␣ homodimers but does not affect DNA binding by T3R␣:RXR␣ heterodimers. We provide evidence that increased activities of T3R␣ at lower T3 concentrations are not due to changes in its T3 binding properties. Instead, the altered response could be mediated by either RXR␣-induced conformational changes, increased stability of heterodimers over homodimers, especially following T3 binding, or both.Thyroid hormone 3,5,3Ј-triiodo-L-thyronine (T3) is required for normal growth and development and maintenance of normal metabolism (for a review, see reference 27). The actions of T3 are mediated through binding to T3 receptors (T3Rs), which belong to the nuclear receptor superfamily of transcription factors encoded by the c-erbA␣ and c-erbA␤ genes (36, 41). Nuclear receptors are ligand-activated transcription factors possessing highly conserved DNA-binding domains and moderately conserved ligand-binding domains (7,15). Together with receptors for retinoic acid (RA) and vitamin D, the T3Rs form a subgroup that recognizes the consensus half-site AGGTCA (7). This sequence can be arranged as a direct repeat, a palindrome, or an inverted repeat, and the spacing between the half sites and their relative orientations determine receptor specificity and the nature of the transcriptional response (26,34,39). For example, unliganded T3R␣ can repress promoters that contain palindromic T3 response elements (T3REs), and this repression is reversed upon binding of T3, resulting in net activation (13, 37). Unliganded T3R␣ can also activate transcription from promoters which contain an inverted repeat of a variant half-site, and this activation is reversed upon T3 binding (34). Whereas the ligand-independent repression of basal promoter activity and the ligand-dependent activation functions are mediated by sequences within the Cterminal ligand-binding domain (4-6, 30, 32), the ligand-independent activation function is medi...

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“…To obtain accurate data, bound [12'I]T3 was determined by filtration using nitrocellulose membranes [19]. The curves demonstrated that, although oleic acid had little effect on the maximum binding capacity of the receptor ( z 0.32 pmol/mg protein), it did alter the dissociation constant from 55 pM to 155 pM; thus, the fatty acid reduced the affinity of the receptor for hormone (Fig.4).…”

Section: Mechanism Of Inhibit Ionmentioning

confidence: 99%

“…Rat liver was homogenized in cold 0.25 M sucrose/5 mM MgCI2/20 mM Tris/HCl, pH 7.6, and centrifuged as described in [19]. The pelleted crude nuclei were washed three times in 0.25 M sucrose/2 mM MgCI2/20 mM Tris/HCl, pH 7.6 (buffer A) containing 0.5% (by vol.)…”

Section: Extraction Of T3 Receptormentioning

confidence: 99%

“…Protein-bound [lZ5I]T3 was determined by filtration of the mixtures on nitrocellulose membranes, under suction at 2 "C. The procedure was described in detail under Standard assay procedure of membrane filtration for thyroid hormone receptors in [19]. Alternatively, bound ['251]T3 was determined in a similar manner to that described in [20]: the reaction mixture was vortexed with AG 1 x 8 resin (24 mg) suspension in cold buffer T (1.0 ml) for 4 s, and left in an ice bath for 4 min.…”

Section: Assay F O R Solubilized Receptormentioning

confidence: 99%

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Unesterified long‐chain fatty acids inhibit thyroid hormone binding to the nuclear receptor

Inoue

Yamamoto

Morisawa

et al. 1989

European Journal of Biochemistry

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Unesterified long-chain fatty acids strongly inhibited thyroid hormone (T3) binding to nuclear receptors extracted from rat liver, kidney, spleen, brain, testis and heart. Oleic acid was the most potent inhibitor, attaining 50% inhibition at 2.8 pM. Oleic acid similarly inhibited the partially purified receptor and enhanced dissociation of the preformed T3-receptor complex. The fatty acid acted in a soluble form and in a competitive manner for the T,-binding sites, thereby reducing the affinity of the receptor for T,. The affinity of the receptor for oleic acid (Ki) was 1.0 pM. In HTC rat hepatoma cells in culture, fatty acids added to the medium reached the nucleus and inhibited nuclear T3 binding; oleic acid being the most potent. T3 binding of the cells was reversibly restored in fresh medium free of added fatty acids. Oleic acid did not affect all the T3-binding sites in the HTC cells: one form (80%) was inhibited and the other was not and these two forms were commonly present in all rat tissues examined. Thus, fatty acids inhibited the solubilized nuclear receptor as well as a class of nuclear T3-binding sites in cells in culture.Specific nuclear receptors for thyroid hormone (triiodothyronine; T3) exist in association with chromatin and participate in the regulation of transcription [l]. T,-responsive elements have been identified in the 5'4anking regions of rat and human growth hormone genes [2 -51. Other examples are the induction by T3 of the or-myosin heavy-chain gene [6, 71, and repression of thyrotropin a-and p-chain genes [8]. The hormone acts also at post-transcriptional steps [9 -111, such as in the stabilization of nuclear mRNA precursors [12]. In addition to the chromatin-associated receptor, other T3-binding sites are present in the soluble cytoplasm, plasma membrane, mitochondria, endoplasmic reticulum and nuclear envelope [33, 141. The chromatin receptor has been separated from other T3 binders, essentially by isolation of nuclei, followed by repeated washing in a solution containing a detergent such as 3-[3-(chloramidopropy1)-dimethylammonio]-1-propane sulfate (Chaps) or Triton X-100, to remove cytoplasmic contaminants and nuclear-envelope-associated binders [14-171.In early work we found that when the solubilized nuclear T3 receptor was treated with a lipase, the T,-binding activity of the receptor was significantly reduced, These events proved to be linked to fatty acids, presumably generated by hydrolysis of lipids present in the solubilized receptor preparation (unpublished observation).In the present study, we show that unesterified long-chain fatty acids inhibited T3 binding by the nuclear receptor solubilized from the chromatin of various rat organs, and also T3 binding in HTC rat hepatoma cells in culture. There were two forms of binding sites, in terms of sensitivity to inhibition in the cultured cells as well as in the isolated nuclei: one was inhibited by fatty acids and designated here as the responder, and the other was not inhibited and was designated the nonresponder. During p...

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Filter-binding assay procedure for thyroid hormone receptors

Cited by 82 publications

References 17 publications

An intramolecular folding sensor for imaging estrogen receptor–ligand interactions

An intramolecular folding sensor for imaging estrogen receptor–ligand interactions

A shift in the Ligand Responsiveness of Thyroid Hormone Receptor α Induced by Heterodimerization with Retinoid X Receptor α

Unesterified long‐chain fatty acids inhibit thyroid hormone binding to the nuclear receptor

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