A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency.

Pit-1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals. Mutations in the gene encoding Pit-1 have been found in two dwarf mouse strains displaying hypoplasia of growth hormone, prolactin, and thyroid-stimulating, hormone-secreting cell types in the anterior pituitary. A point mutation in this gene was identified on one allele in a patient with combined pituitary hormone deficiency. Mutant Pit-1 binds DNA normally but acts as a dominant inhibitor of Pit-1 action in the pituitary.

Evaluating the Hypothalamic-Pituitary-Thyroid (HPT) Axis in Mice.

As the genome of experimental animals has become easier to manipulate, a number of mouse models have been developed to understand in vivo thyroid hormone action. A major site of thyroid hormone action is the HPT axis. While several methods are available that provide a detailed understanding of the HPT axis in mice, many authors choose to include only cursory data about this axis, which can lead to erroneous conclusions about in vivo thyroid hormone action. A standard protocol is proposed to evaluate the HPT axis in mice.

Role of a pituitary-specific transcription factor (pit-1/GHF-1) or a closely related protein in cAMP regulation of human thyrotropin-beta subunit gene expression.

cAMP regulation of the human thyrotropin-beta (TSH beta) gene cAMP was studied in two heterologous cell lines, a human embryonal kidney cell line (293) and a rat pituitary cell line (GH3). In 293 cells, human TSH beta gene expression was not stimulated by the adenylate cyclase activator forskolin or the cAMP analogue 8-bromo-cAMP (8-Br-cAMP). On the other hand, these agents induced human TSH beta gene expression 4-12-fold in GH3 cells. Deletion analysis demonstrated that the regions from +3 to +8 bp and from -128 to -61 bp were both necessary for cAMP stimulation. The latter region contains three DNA sequences homologous to a pituitary-specific transcription factor, Pit-1/GHF-1, DNA-binding site. Gel-mobility assays demonstrated that a radiolabeled human TSH beta probe (-128 to -61 bp) formed five specific DNA-protein complexes with mouse thyrotropic tumor (MTT) nuclear extract and two specific complexes with in vitro translated Pit-1/GHF-1. Four of the five MTT complexes and both in vitro Pit-1/GHF-1 complexes were reduced or eliminated by excess of an unlabeled Pit-1/GHF-1 DNA-binding site from the rat growth hormone gene, but not a mutated version of the same DNA fragment, suggesting that Pit-1/GHF-1 or a closely related thyrotroph protein binds to these DNA sequences. In 293 cells, co-transfection of an expression vector containing the Pit-1/GHF-1 cDNA restored cAMP-responsiveness to the human TSH beta promoter (5.2- and 6.6-fold maximal stimulation by 8-Br-cAMP and forskolin, respectively) but not the herpes virus thymidine kinase promoter (1.2-fold maximal stimulation by either agent). Thus we conclude that the human TSH beta gene is positively regulated by cAMP in GH3 but not 293 cells. Since the human TSH beta gene contains at least one high-affinity binding site for Pit-1/GHF-1 in a region necessary for cAMP stimulation and cAMP stimulation could be restored to the human TSH beta promoter in a previously nonresponsive cell line by the addition of Pit-1/GHF-1, this suggests that Pit-1/GHF-1, or a closely related protein in the thyrotroph, may be a trans-acting factor for cAMP stimulation of the TSH beta gene.

Critical role for thyroid hormone receptor beta2 in the regulation of paraventricular thyrotropin-releasing hormone neurons.

Thyroid hormone thyroxine (T(4)) and tri-iodothyronine (T(3)) production is regulated by feedback inhibition of thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) synthesis in the pituitary and hypothalamus when T(3) binds to thyroid hormone receptors (TRs) interacting with the promoters of the genes for the TSH subunit and TRH. All of the TR isoforms likely participate in the negative regulation of TSH production in vivo, but the identity of the specific TR isoforms that negatively regulate TRH production are less clear. To clarify the role of the TR-beta2 isoform in the regulation of TRH gene expression in the hypothalamic paraventricular nucleus, we examined preprothyrotropin-releasing hormone (prepro-TRH) expression in mice lacking the TR-beta2 isoform under basal conditions, after the induction of hypothyroidism with propylthiouracil, and in response to T(3) administration. Prepro-TRH expression was increased in hypothyroid wild-type mice and markedly suppressed after T(3) administration. In contrast, basal TRH expression was increased in TR-beta2-null mice to levels seen in hypothyroid wild-type mice and did not change significantly in response to induction of hypothyroidism or T(3) treatment. However, the suppression of TRH mRNA expression in response to leptin reduction during fasting was preserved in TR-beta2-null mice. Thus TR-beta2 is the key TR isoform responsible for T(3)-mediated negative-feedback regulation by hypophysiotropic TRH neurons.

Evidence for direct estrogen regulation of the human gonadotropin-releasing hormone gene.

This study is an attempt to determine whether estrogen could directly regulate human gonadotropin-releasing hormone (GnRH) gene expression. Human GnRH expression vectors were constructed by fusing various 5' flanking regions of the human GnRH gene upstream of the luciferase reporter gene (LUC) or the thymidine kinase promoter linked to the chloramphenicol acetyltransferase reporter gene (CAT). These constructs were transiently transfected into a human choriocarcinoma cell line (JEG-3) and LUC or CAT activity was measured after either no treatment or treatment with various concentrations of estradiol. A stimulatory estrogen response element (ERE) was localized to a 32-bp region between -547 and -516 bp. To determine whether estrogen receptor bound to this region of the gene, we performed DNase I footprinting using purified calf uterine estrogen receptor. DNase I footprinting demonstrates a strong footprint between -567 and -514 bp of the human GnRH gene. In addition, an avidin-biotin complex DNA-binding assay demonstrated that a biotinylated DNA fragment containing -541 to -517 bp of the human GnRH gene bound 35S-labeled estrogen receptor as well as a biotinylated DNA fragment containing the xenopus vitellogenin ERE. On the other hand, the negative control biotinylated DNA fragment derived from adenovirus 5 bound insignificant amounts of 35S-labeled estrogen receptor. Both the GnRH ERE and vitellogenin ERE bound 35S-labeled estrogen receptor with high affinity (approximately 1 nM). These data indicate that the human GnRH gene contains an ERE sufficient to mediate a stimulatory response to estrogen in heterologous cells. Based upon these data we hypothesize that the human GnRH gene might also be directly regulated by estrogen in the hypothalamus, and that this regulation may explain the GnRH hypersecretion observed at the time of the preovulatory luteinizing hormone (LH) surge.

Novel insight from transgenic mice into thyroid hormone resistance and the regulation of thyrotropin.

Patients with resistance to thyroid hormone (RTH) exhibit elevated thyroid hormone levels and inappropriate thyrotropin (thyroid-stimulating hormone, or TSH) production. The molecular basis of this disorder resides in the dominant inhibition of endogenous thyroid hormone receptors (TRs) by a mutant receptor. To determine the relative contributions of pituitary versus hypothalamic resistance to the dysregulated production of thyroid hormone in these patients, we developed a transgenic mouse model with pituitary-specific expression of a mutant TR (Delta337T). The equivalent mutation in humans is associated with severe generalized RTH. Transgenic mice developed profound pituitary resistance to thyroid hormone, as demonstrated by markedly elevated baseline and non-triodothyronine (T3)-suppressible serum TSH and pituitary TSH-beta mRNA. Serum thyroxine (T4) levels were only marginally elevated in transgenic mice and thyrotropin-releasing hormone (TRH) gene expression in the paraventricular hypothalamus was downregulated. After TRH administration, T4 concentrations increased markedly in transgenic, but not in wild-type mice. Transgenic mice rendered hypothyroid exhibited a TSH response that was only 30% of the response observed in wild-type animals. These findings indicate that pituitary expression of this mutant TR impairs both T3-mediated suppression and T3-independent activation of TSH production in vivo. The discordance between basal TSH and T4 levels and the reversal with TRH administration demonstrates that resistance at the level of both the thyrotroph and the hypothalamic TRH neurons are required to elevate thyroid hormone levels in patients with RTH.

A circulating, biologically inactive thyrotropin caused by a mutation in the beta subunit gene.

Mutation of a critical carboxy-terminal cysteine residue (C105V) in the thyrotropin-beta (TSH-beta) subunit gene was found in two related families with central hypothyroidism. Affected patients had low thyroid hormone levels and radioactive iodine uptake in the thyroid gland associated with measurable serum TSH. Thyrotropin-releasing hormone-stimulated TSH secretion did not increase thyroid hormone production in these patients as compared to their unaffected siblings, suggesting that the mutant TSH was biologically inactive in vivo. Recombinant TSH harboring this mutation was confirmed to be biologically inactive in an in vitro bioassay. Based on crystallographic structure of chorionic gonadotropin, a disulfide bond between C19 and C105 in the TSH-beta subunit is predicted to form the "buckle" of a "seat belt" that surrounds the common alpha subunit and maintains the conformation and bioactivity of the hormone. This natural mutation of the TSH-beta subunit confirms the importance of the seat belt in the family of pituitary and placental glycoprotein hormones.

A base mutation of the C-erbA beta thyroid hormone receptor in a kindred with generalized thyroid hormone resistance. Molecular heterogeneity in two other kindreds.

Generalized thyroid hormone resistance (GTHR) is a disorder of thyroid hormone action that we have previously shown to be tightly linked to one of the two thyroid hormone receptor genes, c-erbA beta, in a single kindred, A. We now show that in two other kindreds, B and D, with differing phenotypes, there is also linkage between c-erbA beta and GTHR. The combined maximum logarithm of the odds score for all three kindreds at a recombination fraction of 0 was 5.77. In vivo studies had shown a triiodothyronine (T3)-binding affinity abnormality in nuclear receptors of kindred A, and we therefore investigated the defect in c-erbA beta in this kindred by sequencing a major portion of the T3-binding domain in the 3'-region of fibroblast c-erbA beta cDNA and leukocyte c-erbA beta genomic DNA. A base substitution, cytosine to adenine, was found at cDNA position 1643 which altered the proline codon at position 448 to a histidine. By allelic-specific hybridization, this base substitution was found in only one allele of seven affected members, and not found in 10 unaffected members of kindred A, as expected for a dominant disease. Also, this altered base was not found in kindreds B or D, or in 92 random c-erbA beta alleles. These results and the fact that the mutation is predicted to alter the secondary structure of the crucial T3-binding domain of the c-erbA beta receptor suggest this mutation is an excellent candidate for the genetic cause of GTHR in kindred A. Different mutations in the c-erbA beta gene are likely responsible for the variant phenotypes of thyroid hormone resistance in kindreds B and D.

Gene expression of T3-regulated genes in a mouse model of the human thyroid hormone resistance.

AIMS: To understand how thyroid hormone (TH) regulates tissue-specific gene expression in patients with the syndrome of resistance to TH (RTHß), we used a mouse model that replicates the human RTHß, specifically the ∆337T mutation in the thyroid hormone receptor ß (THRß). MAIN METHODS: We investigated the expression of key TH target genes in the pituitary and liver of TRß∆337T and wild type THRß mice by qPCR before and after a T3 suppression test consisting of the administration of increasing concentrations of T3 to hypothyroid mice. KEY FINDINGS: Pituitary Tshb and Cga expression decreased and Gh expression increased in TRß∆337T mice after T3 suppression. The stimulation of positively regulated TH genes was heterogeneous in the liver. Levels of liver Me1 and Thsrp were elevated in TRß∆337T mice after T3 administration. Slc16a2 and Gpd2 did not respond to T3 stimulation in the liver of TRß∆337T mice whereas Dio1 response was lower than that observed in WT mice. Moreover, although Chdh and Upd1 genes were negatively regulated in the liver, the expression of these genes was elevated after T3 suppression. We did not observe significant changes in THRα expression in the liver and pituitary, while THRß levels were diminished in the pituitary and increased in the liver. SIGNIFICANCE: Using a model expressing a THRß unable to bind T3, we showed the expression pattern of liver negative and positive regulated genes by T3.

Familial Congenital Hypothyroidism Caused by Abnormal and Bioinactive TSH due to Mutations in the beta-Subunit Gene.

Hereditary TSH deficiency is a rare autosomal recessive disease described in inbred Japanese families and in Greek and Brazilian kindreds. The TSH-beta-subunit gene has been shown to be the site of mutations that will give rise to truncated proteins that cannot dimerize with the alpha subunit or, alternatively, will produce a mutated TSH that is present in the circulation of the affected patients, but it is biologically inactive. Characteristically, the patients with TSH-beta-subunit-defects are born with congenital hypothyroidism, with very low levels of serum thyroid hormones and serum thyroglobulin and, paradoxically, with serum TSH levels that are consistently undetectable or at very low levels. Goiter is not present at birth, but the low radioactive thyroid uptake will increase after bovine TSH stimulation. Other pituitary hormones responses to provocative tests are normal. The subunit levels are at high concentration and are significantly increased following TRH stimulation. In two kindreds, molecular biological studies have indicated mutations in two different sites of exon 2, generating a peptide that would not dimerize with subunits to synthesize TSH molecules. In one kindred, a truncated TSH-beta protein was translated that generated a biologically inactive but detectable serum TSH molecule. (c) 1997, Elsevier Science Inc. (Trends Endocrinol Metab 1997;8:15-20).

The nuclear corepressors recognize distinct nuclear receptor complexes.

The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms have the capacity to silence gene expression in the absence of their ligands on target response elements. This active repression is mediated by the ability of the corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to recruit a complex containing histone deacetylase activity. Interestingly, NCoR and SMRT share significant differences in the their two nuclear receptor-interacting domains (IDs), suggesting that they may recruit receptors with different affinities. In addition, the role of the receptor complex bound to a response element has not been fully evaluated in its ability to recruit separate corepressors. We demonstrate in this report that the proximal ID in NCoR and SMRT, which share only 23% homology, allows preferential recognition of nuclear receptors, such that TR prefers to recruit NCoR, and RAR prefers to recruit SMRT, to DNA response elements. However, mutations in the TR found in the syndromes of resistance to thyroid hormone can change the corepressor recruited by changing the complex (homodimer or heterodimer) formed on the TRE. These results demonstrate that the corepressor complex recruited can be both nuclear receptor- and receptor complex-specific.

Two separate NCoR (nuclear receptor corepressor) interaction domains mediate corepressor action on thyroid hormone response elements.

The nuclear corepressor (NCoR) binds to the thyroid hormone receptor (TR) in the absence of ligand. NCoR-TR interactions are mediated by two interaction domains in the C-terminal portion of NCoR. Binding of NCoR to TR results in ligand-independent repression on positive thyroid hormone response elements. The interactions between NCoR interaction domains and TR on DNA response elements, however, have not been well characterized. We have found that both interaction domains are capable of binding TR on thyroid hormone response elements. In addition, the NCoR interaction domains interact much more strongly with the TR than those present in the silencing mediator of retinoic acid and TRs (SMRT). Furthermore, deletion of either NCoR interaction domain does not significantly impair ligand-independent effects on positive or negative thyroid hormone response elements. Finally, both NCoR interaction domains appear to preferentially bind TR homodimer over TR-retinoid X receptor heterodimer in electrophoretic mobility shift assays. These data suggest that either NCoR interaction domain is capable of mediating the ligand-independent effects of TR on positive and negative thyroid hormone response elements.

The human thyrotropin-releasing hormone gene is regulated by thyroid hormone through two distinct classes of negative thyroid hormone response elements.

TRH is the principal positive regulator of TSH synthesis and secretion in man. T3 is able to control TRH synthesis through feedback inhibition at the transcriptional level, presumably by binding to its receptor which interacts with one or more negative thyroid hormone response elements (TREs) present within the human TRH promoter. In the present study we have identified the specific negative TREs within the TRH promoter and characterized their ability to interact with thyroid hormone receptors (TRs), and the retinoid X receptor (RXR). Our analysis demonstrates that ligand-independent and dependent regulation of the human TRH promoter is restricted to the TR beta 1 isoform. Deletional analysis of the TRH promoter identified two discrete regions that are responsible for mediating ligand-dependent negative regulation of the TRH promoter. Mutagenesis of potential TR binding half-sites within these regions identified three separate half-sites (site 4 from -55 to -60 base pairs (bp); site 5, +14 to +19 bp; and site 6, +37 to +42 bp) which act in combination to allow for negative regulation. Mutation and/or deletion of each of these sites leads to a loss of negative regulation of the TRH promoter by T3. Gel-mobility shift assays of site 4 and its surrounding nucleotides revealed that this region of the promoter is capable of binding TR monomers, homodimers, and TR-RXR heterodimers. Mutagenesis of site 4 leads to a loss of all binding to this region. The region encompassing sites 5 and 6 binds only TR monomer, and the addition of RXR to the binding reaction leads to a loss of specific monomeric binding. To assess the functional importance of site 4 and its surrounding nucleotides we cotransfected RXR isoforms along with TR beta with TRH promoter constructs containing either site 4 or its mutant. In the presence of wild type site 4 sequence, cotransfected RXR enhanced negative regulation of the TRH promoter. Mutation and or deletion of site 4 leads to a loss of this enhancement. These data demonstrate that two structurally different negative TREs cooperate to allow for negative regulation of the human TRH promoter and that negative regulation is TR isoform-specific and modulated by the RXR-signaling pathway through a novel negative TRE.

A novel mechanism for cyclic adenosine 3',5'-monophosphate regulation of gene expression by CREB-binding protein.

The pituitary-specific transcription factor, Pit-1, is necessary to mediate protein kinase A (PKA) regulation of the GH, PRL, and TSH-beta subunit genes in the pituitary. Since these target genes lack classical cAMP DNA response elements (CREs), the mechanism of this regulation was previously unknown. We show that CREB binding protein (CBP), through two cysteine-histidine rich domains (C/H1 and C/H3), specifically and constitutively interacts with Pit-1 in pituitary cells. Pit-1 and CBP synergistically activate the PRL gene after PKA stimulation in a mechanism requiring both an intact Pit-1 amino-terminal and DNA-binding domain. A CBP construct containing the C/H3 domain [amino acids (aa) 1678-2441], but not one lacking the C/H3 domain (aa 1891-2441), is sufficient to mediate this response. Neither construct augments PKA regulation of CRE-containing promoters. Fusion of either CBP fragment to the GAL4 DNA-binding domain transferred complete PKA regulation to a heterologous promoter. These findings provide a mechanism for CREB-independent regulation of gene expression by cAMP.

Defective retinoic acid regulation of the Pit-1 gene enhancer: a novel mechanism of combined pituitary hormone deficiency.

Pit-1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals. Pit-1 contains two protein domains, termed POU-specific and POU-homeo, which are both necessary for DNA binding and activation of the GH and PRL genes and regulation of the PRL, TSH-beta subunit (TSH-beta), and Pit-1 genes. Pit-1 is also necessary for retinoic acid induction of its own gene during development through a Pit-1-dependent enhancer. Combined pituitary hormone deficiency is caused by defective transactivation of target genes in the anterior pituitary. In the present report, we provide in vivo evidence that retinoic acid induction of the Pit-1 gene can be impaired by a Pit-1 gene mutation, suggesting a new molecular mechanism for combined pituitary hormone deficiency in man.

Isolation and characterization of the human gonadotropin-releasing hormone gene in the hypothalamus and placenta.

The GnRH gene has been cloned in several species, but the location of the promoter and the exact start of transcription have not previously been determined. To characterize the low abundance human GnRH mRNA in the hypothalamus and placenta, we have employed the polymerase chain reaction. The hypothalamus was found to have a 61-base pair first exon, and its transcriptional start site was determined. The human hypothalamic GnRH cDNAs isolated thus far have all contained a short 5' untranslated region which would correspond to this start site. However, all human placental GnRH cDNAs reported to date have a long 5' untranslated region, which extends more than 140-base pairs 5' to this start site in the hypothalamus, suggesting the utilization of an alternative promoter in the placenta. In addition, the human GnRH gene undergoes differential splicing in these tissues. The first intron is removed from the hypothalamic, but retained in the placental, GnRH mRNA. Thus, the placenta has a very long first exon, while the hypothalamus has a comparatively short first exon, followed by a long first intron. This characterization of the human GnRH gene will now allow hormonal regulatory studies to be performed using gene transfer techniques.

Cloning and structure of human genomic DNA and hypothalamic cDNA encoding human prepro thyrotropin-releasing hormone.

The gene encoding human preproTRH was isolated from a human lung fibroblast genomic DNA library with a rat prepro TRH cDNA fragment. The transcriptional unit is 3.3 kilobases in size and contains three exons interrupted by two introns of approximately 1050 and 650 base pairs, respectively. Exon 1 encodes the 5'-untranslated region of the mRNA, exon 2 the putative signal sequence and the initial portion of propeptide, and exon 3 encodes the remainder of the propeptide, which contains six copies of the TRH sequence in contrast to five copies in the rat preproTRH gene. The predicted human preproTRH peptide structure has 242 amino acids compared to 255 amino acids in the rat. Homology with rat preproTRH is 73.3% and 59.5% at the nucleic acid and amino acid levels, respectively. Intron-exon splicing sites and 5' and 3' mRNA borders were confirmed rigorously by sequencing a human preproTRH cDNA using the polymerase chain reaction and human hypothalamic cDNA.

The specificity of interactions between nuclear hormone receptors and corepressors is mediated by distinct amino acid sequences within the interacting domains.

The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms interact with the nuclear corepressors [NCoR (nuclear corepressor protein) and SMRT (silencing mediator for retinoid and thyroid hormone receptors)] in the absence of ligand to silence transcription. NCoR and SMRT contain C-terminal nuclear hormone receptor (NHR) interacting domains that each contain variations of the consensus sequence I/L-x-x-I/V-I (CoRNR box). We have previously demonstrated that TRbeta1 preferentially interacts with NCoR, whereas RARalpha prefers SMRT. Here, we demonstrate that this is due, in part, to the presence of a novel NCoR interacting domain, termed N3, upstream of the previously described domains. An analogous domain is not present in SMRT. This domain is specific for TR and interacts poorly with RAR. Our data suggest that the presence of two corepressor interacting domains are necessary for full interactions with nuclear receptors in cells. Interestingly, mutation of N3 alone specifically decreases binding of NCoR to TR in cells but does not decrease NCoR-RAR interactions. In addition, while the exact CoRNR box sequence of a SMRT interacting domain is critical for recruitment of SMRT by RAR, the CoRNR box sequences themselves do not explain the strong interaction of the N2 domain with TRbeta1. Additional regions distal to the CoRNR box sequence are needed for optimal binding. Thus, through sequence differences in known interacting domains and the presence of a newly identified interacting domain, NCoR is able to preferentially bind TRbeta1. These preferences are likely to be important in corepressor action in vivo.

Tight linkage between the syndrome of generalized thyroid hormone resistance and the human c-erbA beta gene.

Multiple cDNAs belonging to the c-erbA gene family encode proteins that bind T3 with high affinity. However, the biological functions of these multiple thyroid hormone receptors have not yet been clarified. Generalized thyroid hormone resistance (GTHR) refers to a human syndrome characterized by tissue refractoriness to the action of thyroid hormones; several studies have suggested quantitative or qualitative defects in T3 binding to nuclear receptors in certain kindreds. To investigate the biological functions of the c-erbA genes, c-erbA alpha and c-erbA beta, we tested the hypothesis that an abnormal c-erbA gene product is present in GTHR by examining these genes in members of one kindred. Restriction enzyme analysis failed to identify an abnormal pattern in affected individuals suggesting no rearrangements or large deletions. However, we demonstrated that the gene conferring the GTHR phenotype is tightly linked to the c-erbA beta locus on chromosome 3. This linkage strongly suggests that the c-erbA beta gene is important in man as a thyroid hormone receptor and identifies a putative c-erbA beta mutant phenotype with central nervous system, pituitary, liver, metabolic, and growth abnormalities.

Isoform variable action among thyroid hormone receptor mutants provides insight into pituitary resistance to thyroid hormone.

Resistance to thyroid hormone (RTH) is due to mutations in the beta-isoform of the thyroid hormone receptor (TR-beta). The mutant TR interferes with the action of normal TR to cause the clinical syndrome. Selective pituitary resistance to thyroid hormone (PRTH) results in inappropriate TSH secretion and peripheral sensitivity to elevated thyroid hormone levels. Association of the PRTH phenotype with in vitro behavior of the mutant TR has proved elusive. Alternative exon utilization results in two TR-beta isoforms, TR-beta 1 and TR-beta 2, which differ only in their amino termini. Although the TR-beta 1 isoform is ubiquitous, the TR-beta 2 isoform is found predominantly in the anterior pituitary and brain. To date, in vitro evaluation of RTH mutations has focused on the TR-beta 1 isoform. Site-directed mutagenesis was used to create several PRTH (R338L, R338W, V349M, R429Q, I431T) and generalized RTH (delta 337T, P453H) mutations in both TR-beta isoforms. The ability of mutant TRs to act as dominant negative inhibitors of wild type TR-beta function on positive and negative thyroid hormone response elements (pTREs and nTREs, respectively) was evaluated in transient transfection assays. PRTH mutants had no significant dominant negative activity as TR-beta 1 isoforms on pTREs found in peripheral tissues or on nTREs found on genes regulating TSH synthesis. PRTH mutants, in contrast, had strong dominant negative activity on these same nTREs as TR-beta 2 isoforms. Cotransfected retinoid X receptor-alpha was required for negative T3 regulation via the TR-beta 1 isoform but was not necessary for negative regulation via the TR-beta 2 isoform in CV-1 cells. The differing need for retinoid X receptor cotransfection demonstrates two distinct negative T3-regulatory pathways, one mediated by the TR-beta 1 and the other mediated by TR-beta 2. The selective effect of PRTH mutations on the TR-beta 2 isoform found in the hypothalamus and pituitary vs. the TR-beta 1 isoform found in peripheral tissues suggests a molecular mechanism for the PRTH disorder.