Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have cloned the human thyroid hormone receptor beta 1 (hThR beta) from the human breast cancer cell line T47D using the PCR technique. A recombinant baculovirus transfer vector pVL1392/hThR beta was constructed and the full length receptor was expressed in the insect cell line Spodoptera frugiperda (Sf9). Approx. 10-15 x 10(6) receptors are expressed/cell which implies a production level of 2.5-4.0 mg hThR beta/l of cell culture. The expressed hThR beta displayed a single class of binding sites for T3 with high affinity. Western blot analysis using a polyclonal antibody indicated that the molecular weight of the baculovirus expressed receptor is approx. 50 kDa. Crude nuclear extract of hThR beta labeled with [125I]T3 sedimented as a 4 S peak on a glycerol gradient. No receptor could be detected in the cytoplasm indicating its proper translocation to the nuclear compartment. An oligonucleotide containing a palindromic thyroid hormone response element is specifically recognized and retarded in a gel-mobility-shift assay in the presence of nuclear extract of Sf9 cells expressing hThR beta. These data suggest that hThR beta expressed in Sf9 cells is functional and displays characteristics virtually indistinguishable from those of the thyroid hormone receptor (ThR) extracted from mammalian cells. Furthermore, the data indicate that the baculovirus expression system is adequate for large-scale production of receptor for detailed structural and functional studies.
J Steroid Biochem Mol Biol 1991 Jun
PMID:High level expression of functional full length human thyroid hormone receptor beta 1 in insect cells using a recombinant baculovirus. 206 82

The human c-erb A beta gene sequence was inserted in an Escherichia coli expression vector plasmid. The E. coli cells transformed with this plasmid produced proteins with molecular masses of 52 and 50 kDa. These products bound 3,5,3'-triiodo-L-thyronine (T3) with an affinity constant of 4.3 x 10(9) liter/mol. The order of affinity for iodothyronine analogs was triiodothyroacetic acid greater than T3 greater than 3,5,3'-triiodo-D-thyronine greater than L-thyroxine. Affinity labeling experiments showed that the 50 kDa protein was covalently labeled with [125I]T3, and this was competed by triiodothyroacetic acid, T3, and L-thyroxine (from potent to weaker competitor). The c-erb A protein bound to calf thymus DNA-cellulose and the binding was inhibited by 0.3 M KCl or 10 mM pyridoxal 5'-phosphate. Aqueous two-phase partitioning studies showed that the c-erb A product became less hydrophobic upon T3 or triiodothyroacetic acid binding. The same finding was obtained when T3 bound to partially purified rat liver nuclear thyroid hormone receptor. However, thyroxine binding globulin became more hydrophobic upon T3 binding. Since the T3 molecule partitioned preferentially into the upper polyethylene glycol-rich phase, the alteration of partitioning behavior of thyroxine binding globulin was explained by a simple additive effect of T3. In contrast, the alteration of partitioning behavior of the c-erb A product or receptor reflected a conformational transition upon T3 binding. The c-erb A protein expressed in E. coli showed various characteristics similar to classical thyroid hormone receptor and may be useful in studying the structure and function of the thyroid hormone receptor.
Mol Cell Endocrinol 1990 Apr 17
PMID:Human c-erb A protein expressed in Escherichia coli: changes in hydrophobicity upon thyroid hormone binding. 216 65

We studied the functional significance of a region of the thyroid hormone receptor ligand binding domain which is conserved in all members of the erbA superfamily. The homologous region of the glucocorticoid receptor has been implicated in the binding of heat shock protein 90, and deletions of the glucocorticoid receptor that include this region result in constitutive activity. Both deletion and point mutations were made in this area of the rat beta 1 thyroid hormone receptor (amino acids 286-305), and the functional consequences of these mutations were analyzed in JEG cells by transient cotransfection along with a T3 responsive reporter gene. All mutations studied resulted in significant inhibition of ligand-dependent transcriptional regulation without inducing significant constitutive activity. For some mutations, the lack of transcriptional response correlated with a diminished ability to bind ligand. However, point mutations of amino acids 288, 290, and 300 resulted in impaired transcriptional regulation despite wild type T3 binding affinity. In addition, mutations of these three amino acids failed to impair localization of the receptor to the nucleus or binding to DNA in vitro. Cotransfection of plasmids expressing the wild type and mutant T3 receptor proteins resulted in inhibition of the wild type T3 receptor function. We conclude that this region of the rat beta 1 T3 receptor is essential for full transcriptional activity, but this is not due to a role in T3 binding, DNA binding, or nuclear localization. We postulate that nuclear factors may need to bind to this region for full transcriptional activity.
Mol Endocrinol 1990 May
PMID:Mutational analysis identifies a new functional domain of the thyroid hormone receptor. 227 55

Multiple forms of human thyroid hormone (T3) receptor have been identified, including true receptors that bind T3 (alpha 1 and beta) and a splicing variant (alpha 2) that does not bind T3. The alpha 1- and beta-receptors activate transcription through interactions with positive thyroid response elements (TREs). The alpha 2 variant is unable to activate transcription and has been reported to inhibit alpha 1 or beta stimulation of positive TREs, a property referred to as dominant negative activity. In this report we have performed studies to assess the functional properties of different members of the thyroid receptor family with regard to both positive and negative transcriptional regulation. The alpha 1-, alpha 2-, and beta-receptors were each coexpressed in JEG-3 cells with either TreTKCAT (CAT = chloramphenicol acetyltransferase), a reporter gene that contains a positive TRE, or TSH alpha CAT, a negatively regulated reporter gene. The alpha 1 and beta isoforms stimulated transcription of TreTKCAT and inhibited TSH alpha CAT transcription in a T3-dependent manner, whereas the alpha 2 variant was inactive. When coexpressed with alpha 1- or beta-receptors, alpha 2 inhibited regulation of positive TREs, but the effects of alpha 2 were modest and only occurred when relatively high doses of receptor were transfected. The alpha 2-receptor variant did not affect negative regulation by alpha 1- or beta-receptors. Thus, in both positive and negative regulation, thyroid hormone receptor isoforms that bind T3 (alpha 1, beta) are functional, whereas the alpha 2 isoform, which does not bind T3, is not functional.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1990 Oct
PMID:Negative and positive transcriptional regulation by thyroid hormone receptor isoforms. 228

We have expressed three forms of human thyroid hormone receptor (hTR alpha 1, alpha 2, and beta) in cultured cells by transient transfection. hTR alpha 1 and beta transfected cells showed increased triiodothyronine (T3) binding capacity, but hTR alpha 2 transfected cells did not. When hTR alpha 1 or beta was cotransfected with pUrGH(S), in which a portion of the rat GH 5' flanking region (-236/-147) was ligated into the CAT reporter plasmid (pUTKAT1), T3 increased CAT gene expression. When hTR alpha 2 was cotransfected with pUrGH(S), T3 did not alter CAT gene expression. When hTR alpha 1 or beta was cotransfected with pUrGH(O), in which a synthetic oligonucleotide representing the TRE from the rat GH 5' flanking region (-189/-160) was substituted for the natural enhancer in pUTKAT1, T3 increased CAT gene expression. When hTR alpha 1 and beta were cotransfected with pUrGH(O), induction by T3 was increased. When hTR alpha 2 was cotransfected with hTR alpha 1 or beta, induction by T3 was decreased. These results indicate that hTR alpha 1 and beta function as native TR, that hTR alpha 1 and beta can recognize the same TRE, that hTR alpha 1 and beta can function additively, and that hTR alpha 2 can inhibit the action of hTR alpha 1 and beta.
Mol Cell Endocrinol 1990 Sep 10
PMID:The roles of three forms of human thyroid hormone receptor in gene regulation. 228 27

A cDNA that encodes a third type of human thyroid hormone receptor (hTR alpha 1) has been isolated from a skeletal muscle library. The cDNA encodes a 410 amino acid protein, Mr = 46,820. When expressed and translated in vitro, hTR alpha 1 binds T3 with an association constant (ka) of 1.8 x 10(9) M-1. Comparison of the DNA sequence of hTR alpha 1 and a previously identified alpha type thyroid hormone receptor (hTR alpha 2) suggests that they could be transcribed from the same gene, and that alternative RNA splicing results in the synthesis of either hTR alpha 1 or hTR alpha 2. Two mRNA (3.2 kilobases and 6 kilobases) of hTR alpha 1 have been detected in several tissues. At least three types of thyroid hormone receptors (hTR alpha 1, alpha 2, beta), which possess similar affinities for hormone ligands, can be expressed in the same tissue.
Mol Endocrinol 1988 Nov
PMID:Characterization of a third human thyroid hormone receptor coexpressed with other thyroid hormone receptors in several tissues. 246 49

The NGFI-B cDNA was previously isolated by virtue of its induction by nerve growth factor (NGF) in PC12 cells. It encodes a 61-kilodalton protein that has two regions of extensive homology with members of the steroid/thyroid hormone receptor gene family. The rat NGFI-B gene is approximately 7.6 kilobases long and is interrupted by six introns. Although the exon-intron structure of the gene is similar to those of several other members of the steroid/thyroid hormone receptor gene family, there is a novel splice site within the DNA-binding domain which suggests that NGFI-B constitutes yet another evolutionary digression from a postulated common ancestral receptor gene. Primer extension and S1 nuclease protection assays were used to determine the transcription initiation site, which displayed the heterogeneity typical of genes that lack a TATA box. Sequence analysis of the 5' flanking region revealed several GC boxes but no identifiable TATA box. Four potential AP1 binding sites were identified at nucleotides -49, -78, -222, and -242. Neither the serum response element nor the CArG box element, two sequences found in other growth factor-inducible genes, was detected in this region of the growth factor-inducible NGFI-B gene. Nevertheless, results of nuclear runoff experiments demonstrated that the NGFI-B gene was transcriptionally activated by nerve growth factor in PC12 cells. In vivo, a rapid, dramatic increase in NGFI-B mRNA was observed in the cerebral cortex, midbrain, and cerebellum of animals that experienced a convulsant-induced seizure.
Mol Cell Biol 1989 Oct
PMID:The NGFI-B gene, a transcriptionally inducible member of the steroid receptor gene superfamily: genomic structure and expression in rat brain after seizure induction. 247 23

A cDNA encoding a novel member of the thyroid/steroid hormone receptor superfamily, called Rev-ErbA alpha, has been isolated from a rat GH3 cell library. Rev-ErbA alpha is an approximately 56-kilodalton protein most similar in structure to the thyroid hormone receptor (c-erbA) and the retinoic acid receptor, but it does not bind either thyroid hormone or retinoic acid. The mRNA encoding Rev-ErbA alpha is present in many tissues and is particularly abundant in skeletal muscle and brown fat. A genomic DNA fragment containing the entire Rev-ErbA alpha cDNA sequence was isolated and characterized. Remarkably, this DNA fragment also contained a portion of the c-erbA alpha gene. r-erbA alpha-1 and r-erbA alpha-2 are alternative splice products of the c-erbA alpha gene and are members of the receptor superfamily. The genes encoding Rev-ErbA alpha and r-erbA alpha-2 overlap, with their coding strands oriented opposite one another. A 269-base-pair segment of the bidirectionally transcribed region is exonic in both the Rev-ErbA alpha and r-erbA alpha-2 genes, resulting in complementary mRNAs. Thus, through alternative splicing and opposite-strand transcription, a single genomic locus codes for three different members of the thyroid/steroid hormone receptor superfamily. Potential implications of this unusual genomic arrangement are discussed.
Mol Cell Biol 1989 Mar
PMID:A novel member of the thyroid/steroid hormone receptor family is encoded by the opposite strand of the rat c-erbA alpha transcriptional unit. 254 65

The thyroid hormones and retinoic acid are potent modulators of differentiation, development, and gene expression. The transcriptional activities of these ligands are mediated by closely related nuclear receptors which bind and activate identical hormone responsive DNA elements. We noticed that a region within the ligand binding or E domain is well conserved between receptors for these hormones. This region contains hydrophobic heptad repeats that are structurally similar to the leucine-zipper dimerization domain. To study the function of this conserved domain, we examined the transcriptional responses of thyroid hormone receptor/c-erbA deletion mutants which lacked the heptad repeats. We previously reported that the chick c-erbA-alpha possesses hormone-independent (constitutive) activity in cells which express endogenous rat thyroid hormone receptor. We now demonstrate that this activity is abolished upon deletion of the conserved heptad repeats. This suggests that the heptad repeats mediate in vivo interactions between chick c-erbA and rat thyroid hormone receptors. To further test this hypothesis deletion mutants of chick c-erbA were constructed which contained all eight heptad repeats but which lacked the zinc-finger DNA binding domain. Although these mutants are transcriptionally inactive, they act in a dominant-negative fashion to block trans-activation by both the chick c-erbA-alpha and the endogenous thyroid hormone and retinoic acid receptors. We suggest that the heptad repeats mediate the formation of inactive mutant/wild-type hetero-dimers. Dimer formation suggests a mechanism to account for the dominant-negative phenotypes displayed by nonhormone binding variants of c-erbA, the proto-oncoprotein v-erbA and patients with the generalized thyroid hormone resistance syndrome.
Mol Endocrinol 1989 Oct
PMID:A domain containing leucine-zipper-like motifs mediate novel in vivo interactions between the thyroid hormone and retinoic acid receptors. 255 97

At least two thyroid hormone receptor (hTR) genes are present in humans, but the significance of this multiplicity is unknown. These receptors could have differences in tissue distribution or possess different functions. We studied the distribution and abundance of three hTR mRNAs (hTR beta, hTR alpha 1, and hTR alpha 2) by Northern blot analysis. Three mRNAs were expressed in all tissues examined. hTR beta was strongly expressed in brain and prostate predominantly as a 10.0-kilobase (kb) mRNA. This mRNA was also expressed in thyroid and was much less abundant in liver, kidney, placenta, tonsil, and spleen. hTR alpha 1 is represented by two mRNAs with sizes of 6.0 and 3.2 kb. The 6.0-kb mRNA was constantly less abundant than the 3.2-kb mRNA. hTR alpha 2 was detected as a single mRNA with a size of 3.2 kb, using a probe unique for this mRNA. Both hTR alpha 1 and hTR alpha 2 were strongly expressed in brain, prostate, and thyroid and much less in other tissues. The relative amounts of the three hTR mRNAs were roughly parallel in each tissue. It is of interest that none of these hTRs was abundant in liver, which is the major thyroid hormone-responsive organ. Another hTR may be present in liver.
Mol Endocrinol 1989 Feb
PMID:Expression of three forms of thyroid hormone receptor in human tissues. 271 Jan 39


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>