A homozygous deletion in the c-erbA beta thyroid hormone receptor gene in a patient with generalized thyroid hormone resistance: isolation and characterization of the mutant receptor.

Different point mutations have been identified in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene that are associated with variant phenotypes of generalized thyroid hormone resistance (GTHR). In most cases of GTHR, heterozygotes are affected; a single mutant allele results in the inhibition of the function of normal thyroid hormone receptors. We report here a novel genetic abnormality, a 3-basepair (bp) deletion in the T3-binding domain of the beta-receptor in a kindred, S, with GTHR. One patient, S1, was the product of a consanguineous union of two heterozygotes and was homozygous for this defect. Heterozygotes from kindred S harbored a CAC deletion at nucleotides 1295-1297, which resulted in the deduced loss of amino acid residue threonine at codon 332, and they displayed elevated free T4 levels and inappropriately normal TSH levels characteristic of other kindreds with GTHR. However, patient S1, who had two mutant alleles, had markedly elevated TSH and free T4 levels and displayed profound abnormalities in brain development and linear growth. A fibroblast c-erbA beta cDNA extending from codon 175 to stop codon 457 was cloned from patient S1, sequenced, and used to create a full-length mutant cDNA. The kindred S mutant receptor was synthesized in vitro and did not bind T3. This mutant receptor did bind with similar avidity as the wild-type human beta-receptor to thyroid hormone response elements of the human TSH beta (-12 to 43 bp) and rat GH (-188 to -160 bp) genes. Kindred S showed the effect in man of heterozygous and homozygous expression of a dominant negative form of c-erbA beta.     

An essential role of domain D in the hormone-binding activity of human beta 1 thyroid hormone nuclear receptor

By analogy with steroid receptors, human placental thyroid hormone nuclear receptor (hTR beta 1) could be divided into four functional domains: A/B (Met1-Leu101), C (Cys102-Ala170), D (Thr171-Lys237), and E (Arg238-Asp456). The E domain was thought to bind thyroid hormone. To evaluate whether domain E alone is sufficient to bind T3 or requires the presence of other domains for functional T3-binding activity, a series of deletion mutants was constructed. The mutants were expressed in Escherichia coli, and the expressed proteins were purified. Analysis of the T3-binding affinity and analog specificity of the purified truncated hTR beta 1 indicated that domain E alone did not have T3-binding activity. Extension of the amino-terminal sequence of domain E to include part of domain D yielded a mutant (Lys201-Asp456) with a Ka for T3 of 0.5 +/- 0.2 x 10(9) M-1. Further extension to include the entire domain D (Met169-Asp456) yielded a mutant with T3-binding activity with a Ka of 0.8 +/- 0.1 x 10(9) M-1. Further extension of the amino-terminal sequence to include domain C increased the affinity for T3 by nearly 2-fold (Ka = 1.5 +/- 0.4 x 10(9) M-1). The Ka for the wild-type hTR beta 1 is 1.5 +/- 0.2 x 10(9) M-1. Furthermore, mutant (Met169-Asp456) binds to 3',5',3-triiodo-L-thyropropionic acid, D-T3, L-T4, and L-T3 with 307%, 37%, 7%, and 0.1%, respectively, of the activity of L-T3. This order of analog affinity is similar to that of the wild-type hTR beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

The thyroid hormone receptors: molecular basis of thyroid hormone resistance.

Major progress has been achieved in the mechanism of action of thyroid hormones thanks to the identification of the T3 receptor as the product of the proto-oncogene c-erbA. Recognition of subsets of receptors with and without T3-binding properties and of the interaction of different receptors with each other leads to new insights in cell regulation and development. In thyroid hormone resistance, distinct mutations in the T3-binding domain of thyroid hormone receptor (TR)beta have been identified in unrelated families. No correlation between the type of mutation and tissue resistance has been established. Mutant TRs bind to thyroid hormone response elements (TREs) on both negative or positive T3-controlled genes. Subjects with heterozygous TR beta gene deletion are not affected, supporting the hypothesis that mutant TRs act through a dominant negative effect. In generalized thyroid hormone resistance, mutated TR beta may interfere through competition for TREs and/or formation of inactive dimers. Finally, deficiency in T3 receptor auxiliary protein or other accessory proteins or competition between mutant and normal TRs for these factors is not excluded.     

Characterization of a third human thyroid hormone receptor coexpressed with other thyroid hormone receptors in several tissues

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.     

A point mutation (Ala229 to Thr) in the hinge domain of the c-erbA beta thyroid hormone receptor gene in a family with generalized thyroid hormone resistance.

Various point mutations in the c-erbA thyroid hormone receptor (TR) beta gene of unrelated kindreds have been reported to be responsible for different phenotypes of generalized thyroid hormone resistance. We now report a new point mutation, Td, in one of two TR beta alleles of three affected members of one family, designated family T. In contrast to the previously described point mutations, all located in the T3-binding domain of the TR beta gene, mutation Td was identified in the carboxy-terminal part of the hinge domain. Direct sequencing of the polymerase chain reaction-amplified whole coding region of the patients' fibroblast TR beta genes displayed a single guanine to adenine transition at cDNA nucleotide position 985. This altered alanine (GCC) to threonine (ACC) in codon 229. Garnier prediction of the consequence of the mutation indicated an altered secondary structure. The G----A nucleotide substitution was not present in 80 random TR beta alleles, suggesting that this point mutation is responsible for generalized thyroid hormone resistance in family T. The in vitro expressed mutant TR beta was shown to bind with high affinity to various thyroid hormone response elements. However, the affinity of the TR beta to bind to T3 was reduced 3-fold, indicating that the hinge domain of the TR beta is important for full ligand-binding activity. Moreover, it seems that multiple subdomains of the TR beta interact cooperatively to achieve optimal T3 activity.     

Differences in antibody recognition of the triiodothyronine nuclear receptor and c-erbA products

The in vitro translated products of several c-erbA cDNAs have recently been shown to bind thyroid hormones with high affinity and have been termed thyroid hormone receptors. We have used a panel of five erbA-related antibodies to probe the relationship between c-erbA translated products and thyroid hormone receptors, as conventionally measured by 125I-T3 labeling of nuclear extracts. All five antibodies immunoprecipitated the chick c-erbA translated products, but only one of them recognized chick liver and brain T3 receptor, as judged by acceleration of sedimentation through sucrose gradients. None of the antibodies reacted with rat liver and brain or human liver T3 receptors, although one antibody did immunoprecipitate a human c-erbA translated product. We conclude that the T3 receptor, as conventionally measured from these sources, is related but not identical to recently cloned c-erbA sequences.     

Purification and partial characterization of a nonprimate growth hormone receptor.

Pregnant rabbit liver membranes have been shown to possess two types of receptors by displacement analysis, a growth hormone (GH) receptor which binds bovine growth hormone with an affinity constant (KA) of 3 x 10(9) M-1 and ovine prolactin with a KA of 3 x 10(8) M-1, and a prolactin (Prl)-specific receptor which binds ovine prolactin with a KA of 5 x 10(9) M-1. The prolactin-specific receptor when solubilized with Triton exhibits a 4-fold increase in the its KA while the KA of the growth hormone receptor decreases slightly to 2 x 10(9) M-1 after solubilization. The 10-fold difference in affinity which results has been exploited to facilitate the separation of these two receptors by differential affinity chromatography on human growth hormone (hGH) affinity gels. The growth hormone receptor is eluted from the gel with 4 M urea while 5 M MgCl2 is required to elute the prolactin receptor. Conditions of affinity chromatography have been optimized, and further purification of the GH receptor by preparative isoelectric focusing and Sepharose 6B gel filtration resulted in a more than 8000-fold purification of the receptor. This material had a Stokes radius of 62 A, consistent with a molecular weight of 300,000 and gave one main band (75,000 to 80,000) and two minor bands on sodium dodecyl sulfate (SDS) polyacrylamide gels, which could be interpreted as indicating a tetrameric receptor. The GH receptor was shown to be a sialoglycoprotein (or closely associated with sialoglycoprotein) by analytical isoelectric focusing with an isoelectric point of 4.6. Specificity studies with the highly purified receptor confirmed the initial hypothesis that this receptor is capable of binding bovine growth hormone (bGH) with high affinity and ovine prolactin (oPrl) with low affinity, in contrast to the prolactin-specific receptor.     

Alpha and beta thyroid hormone receptors bind immediately adjacent to the rat growth hormone gene TATA box in a negatively hormone-responsive promoter region

We report that alpha and beta type rat thyroid hormone receptors bind specifically and with high affinity to the 10-base pair sequence immediately 3' of the rat growth hormone TATA box (positions -25 to -16) in a region of the rat growth hormone promoter which can be negatively hormone responsive (nTRE). The receptors have approximately 7-fold lower affinity in vitro for the nTRE than for the thyroid hormone-responsive enhancer of the rat growth hormone gene (TRE). Proteins extracted with high salt concentration from rat pituitary cell nuclei enhance binding of the receptors to both the TRE and nTRE. A modification of the avidin-biotin complex DNA binding assay which enhances the sensitivity of the assay approximately 100-fold was used in these studies. The immediate proximity of a receptor binding site to the rat growth hormone TATA box suggests that direct interaction between receptor and TFIID (the TATA binding protein) mediates nTRE activity.     

A detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human thyrotropin beta-subunit gene.

The first exon of the human thyrotropin-beta (hTSH beta) gene has been demonstrated in our laboratory to contain a major thyroid hormone inhibitory element. In order to characterize fully this element, we have performed a detailed functional and structural scanning mutational analysis of this element. Various -1192 to +37 (base pairs) bp fragments of the hTSH beta gene containing consecutive five deoxythymidine substitution mutations of the first exon were inserted into a luciferase reporter plasmid and transiently transfected into human embryonal cells (293) and stably transfected into rat pituitary cells (GH3). Two domains (domain 1 and 2) were identified by scanning mutations that were essential for function of the thyroid hormone inhibitory element: +3 to +13 bp and +28 to +37 bp. Biotinylated DNA fragments containing -12 to +43 bp of the hTSH beta gene and the identical scanning mutations demonstrate that in vitro synthesized c-erbA-beta binding is disrupted as much as 95% by mutations from -3 to +17 bp and to a lesser extent (20-30%) by mutations from +23 to +27 bp and from +33 to +43 bp. Domain 1 displayed a higher affinity for c-erbA-beta than domain 2 in avidin-biotin complex DNA-binding and gel-mobility assays. Using increasing amounts of in vitro synthesized c-erbA-beta, we were unable to demonstrate more than one protein-DNA complex in gel-mobility assays. However, using the avidin-biotin complex DNA-binding assay and the cross-linking reagent, 1,6-bismaleimidohexane, we were able to demonstrate thyroid hormone receptor dimer formation on domain 1 but not to any significant extent on domain 2. In conclusion, functional and DNA-binding studies suggest that the thyroid hormone receptor binds to two distinct regions in the first exon of the hTSH beta gene. The upstream site (domain 1) binds c-erbA-beta with higher affinity and is capable of binding c-erbA-beta as a dimer under some conditions, while the downstream site (domain 2) appears to bind a single molecule of c-erbA-beta with lower affinity. These results suggest that thyroid hormone receptor, binding to at least two sites in the first exon, act in conjunction to mediate T3 inhibition of hTSH beta expression.     

Functional properties of human thyroid hormone receptor beta 1 overexpressed using baculovirus

We have overexpressed the human beta 1 thyroid hormone receptor in insect cells using a recombinant baculovirus to a level of 5-10% of total cellular protein. The recombinant protein migrates as a 50 kDa band by SDS-PAGE and Western blot analysis. The expressed receptor binds to L-T3 with a Kd of 1.3 +/- 0.4 x 10(-10) M and to thyroid hormone analogues with an affinity hierarchy of TRIAC greater than L-T3 greater than L-T4 greater than rT3. Gel retardation assays show highly specific receptor binding to a TRE which is modified by the presence of ligand and avidin-biotin complex DNA analysis shows a Kd of 6.2 +/- 2.0 x 10(-10) M for this interaction. These results indicate high level expression of hTR beta with authentic hormone and DNA binding properties.     

Hormone and DNA binding activity of a purified human thyroid hormone nuclear receptor expressed in Escherichia coli

Using a T7 expression system, large amounts of the human placental c-erbA protein (h-TR beta 1) were expressed. From 1 liter of Escherichia coli culture, approximately 50-100 micrograms of purified h-TR beta 1 were obtained. Analysis of the binding data indicated that the purified h-TR beta 1 binds to 3,3',5-triiodo-L-thyronine (T3) with a Ka = 2.8 x 10(9) M-1. It binds to 3,3',5-triiodo-L-thyropropionic acid, 3,3',5-triiodo-L-thyroacetic acid, D-T3, L-thyroxine (T4), and 3',5',3-triiodo-L-thyronine with 475, 120, 39, 7, and 0.1%, respectively, of the activity of L-T3. This order of binding activity to T3 analogs is similar to that reported for the T3 nuclear receptor identified in tissues or cultured cells. Furthermore, the purified h-TR beta 1 binds to the T3 response element of the rat growth hormone gene. Thus, the purified h-TR beta 1 is active. To identify the hormone binding domain, the purified h-TR beta 1 was affinity labeled with underivatized [3',5'-125I]T4. A partial digestion by trypsin yielded a 125I-labeled 25-kDa fragment which was identified to be the domain Phe240-Asp456 by amino acid sequencing. Thus, the purified h-TR beta 1 appears suitable for other structural and functional studies.