Reduced Transactivation Activity Research Articles

A Novel Inborn Error in the Ligand-Binding Domain of the Vitamin D Receptor Causes Hereditary Vitamin D-Resistant Rickets

Mutations in the vitamin D receptor (VDR) cause hereditary vitamin D-resistant rickets (HVDRR), an autosomal recessive disease resulting in target organ resistance to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In this report, we describe the clinical case and molecular basis of HVDRR in an Asian boy exhibiting the typical clinical features of the disease including alopecia. Using cultured dermal fibroblasts from the patient, 1,25(OH)2D3 resistance was demonstrated by a shift in the dose response required for 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) mRNA induction. Western blot showed that the cells express a normal size VDR but contained reduced levels of receptor compared to normal cells. At 24°C, the affinity of the patient's VDR for [3H]1,25(OH)2D3 was 50-fold lower than the VDR in normal fibroblasts. Sequence analysis identified a unique T to G missense mutation in exon 6 that changed phenylalanine to cysteine at amino acid 251 (F251C). The recreated F251C mutant VDR showed reduced transactivation activity using a 24-hydroxylase promoter-luciferase reporter. Maximal transactivation activity exhibited by the WT VDR was not achieved by the mutant VDR even when the cells were treated with up to 10−6 M 1,25(OH)2D3. However, the transactivation activity was partially rescued by addition of RXRα. In the yeast two-hybrid system and GST-pull-down assays, high concentrations of 1,25(OH)2D3 were needed to promote F251C mutant VDR binding to RXRα, indicating defective heterodimerization. In conclusion, a novel mutation was identified in the VDR LBD that reduces VDR abundance and its affinity for 1,25(OH)2D3 and interferes with RXRα heterodimerization resulting in the syndrome of HVDRR.

Murine RAR?4 displays reduced transactivation activity, lower affinity for retinoic acid, and no anti-AP1 activity

The biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, beta, and gamma) and retinoid X receptors (RXR alpha, beta, and gamma). Each of the RARs is expressed as four to seven different isoforms. Four isoforms of RAR beta (beta1, beta2, beta3, and beta4), which differ only in their N-terminal sequence (A domain) have been described. These RARbeta isoforms display a specific pattern of expression in developing and adult animals and are highly evolutionarily conserved suggesting that they mediate distinct cellular effects of vitamin A. Experiments were performed to examine directly the RA-binding activity, transactivation activity, and anti-AP1 activity of each of these four RARbeta isoforms. The results demonstrate that RARbeta1, beta2, and beta3 bind RA with a similar K(d) value, have a similar EC(50) value in RA-dependent transactivation assays and inhibit AP1 activity to a similar level. By contrast, RARbeta4 has an elevated K(d) for RA, an increased EC(50) value in RA-dependent transactivation assays and does not display the ability to inhibit AP1 activity. This provides additional evidence that at least one RAR isoform, RARbeta4, may mediate distinct activities within a cell. Furthermore, these data suggest that the presence of an A domain in RARbeta is important for modulating these activities of RARs.

Murine RARβ4 displays reduced transactivation activity, lower affinity for retinoic acid, and no anti-AP1 activity

The biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARα, β, and γ) and retinoid X receptors (RXR α, β, and γ). Each of the RARs is expressed as four to seven different isoforms. Four isoforms of RAR β (β1, β2, β3, and β4), which differ only in their N-terminal sequence (A domain) have been described. These RARβ isoforms display a specific pattern of expression in developing and adult animals and are highly evolutionarily conserved suggesting that they mediate distinct cellular effects of vitamin A. Experiments were performed to examine directly the RA-binding activity, transactivation activity, and anti-AP1 activity of each of these four RARβ isoforms. The results demonstrate that RARβ1, β2, and β3 bind RA with a similar Kd value, have a similar EC50 value in RA-dependent transactivation assays and inhibit AP1 activity to a similar level. By contrast, RARβ4 has an elevated Kd for RA, an increased EC50 value in RA-dependent transactivation assays and does not display the ability to inhibit AP1 activity. This provides additional evidence that at least one RAR isoform, RARβ4, may mediate distinct activities within a cell. Furthermore, these data suggest that the presence of an A domain in RARβ is important for modulating these activities of RARs. J. Cell. Biochem. 77:604–614, 2000. © 2000 Wiley-Liss, Inc.

A missense mutation in hepatocyte nuclear factor-4 alpha, resulting in a reduced transactivation activity, in human late-onset non-insulin-dependent diabetes mellitus.

Non-insulin-dependent diabetes mellitus (NIDDM) is a heterogeneous disorder characterized by hyperglycemia resulting from defects in insulin secretion and action. Recent studies have found mutations in the hepatocyte nuclear factor-4 alpha gene (HNF-4alpha) in families with maturity-onset diabetes of the young (MODY), an autosomal dominant form of diabetes characterized by early age at onset and a defect in glucose-stimulated insulin secretion. During the course of our search for susceptibility genes contributing to the more common late-onset NIDDM forms, we observed nominal evidence for linkage between NIDDM and markers in the region of the HNF-4alpha/MODY1 locus in a subset of French families with NIDDM diagnosed before 45 yr of age. Thus, we screened these families for mutations in the HNF-4alpha gene. We found a missense mutation, resulting in a valine-to-isoleucine substitution at codon 393 in a single family. This mutation cosegregated with diabetes and impaired insulin secretion, and was not present in 119 control subjects. Expression studies showed that this conservative substitution is associated with a marked reduction of transactivation activity, a result consistent with this mutation contributing to the insulin secretory defect observed in this family.

Identification of amino acids in the tau 2-region of the mouse glucocorticoid receptor that contribute to hormone binding and transcriptional activation.

The tau 2-region of steroid hormone receptors is a highly conserved region located at the extreme N-terminal end of the hormone-binding domain. A protein fragment encoding tau 2 has been shown to function as an independent transcriptional activation domain; however, because this region is essential for hormone binding, it has been difficult to determine whether the tau 2-region also contributes to the transactivation function of intact steroid receptors. In this study a series of amino acid substitutions were engineered at conserved positions in the tau 2-region of the mouse glucocorticoid receptor (mGR, amino acids 533-562) to map specific amino acid residues that contribute to the hormone-binding function, transcriptional activation, or both. Substitution of alanine or glycine for some amino acids (mutations E546G, P547A, and D555A) reduced or eliminated hormone binding, but the transactivation function of the intact GR and/or the minimum tau 2-fragment was unaffected for each of these mutants. Substitution of alanine for amino acid S561 reduced transactivation activity in the intact GR and the minimum tau 2-fragment but had no effect on hormone binding. The single mutation L550A and the double amino acid substitution L541G+L542G affected both hormone binding and transactivation. The fact that the S561A and L550A substitutions each caused a loss of transactivation activity in the minimum tau 2-fragment and the full-length GR indicated that the tau 2-region does contribute to the overall transactivation function of the full-length GR. Overall, the N-terminal portion of the tau 2-region (mGR 541-547) was primarily involved in hormone binding, whereas the C-terminal portion of the tau 2-region (mGR 548-561) was primarily involved in transactivation.

Single Amino Acid Change in Tat Determines the Different Rates of Replication of Two Sequential HIV-1 Isolates

Human immunodeficiency virus type 1 (HIV-1) isolates display differences in their patterns of replication in vitro. Previous studies with recombinant viruses generated between two sequential HIV-1 isolates that differ in replication rates have shown that the determinant for this biological property maps to a region of the viral genome that encompasses the tat gene. In the present study, we examined if there is a functional difference in the Tat protein of the two isolates. We observed that the level of transactivation induced by Tat of the fast replicating, highly cytopathic virus (HIV-1 SF13mc) was three- to eightfold higher than the level seen by the Tat of the slow replicating virus (HIV-1 SF2mc). Furthermore, a single amino acid mutation in the HIV-1 SF13mc Tat leads to reduced transactivation activity of the mutant protein and a delayed replication rate of the mutant virus. Thus, selection of a Tat variant with higher transactivation potential could be responsible for the increased virus production that is often associated with a pathogenic HIV strain.

Structural analysis of wild-type and mutant human immunodeficiency virus type 1 Tat proteins

We expressed the human immunodeficiency virus type 1 transactivator protein, Tat, in the wheat germ cell-free translation system and found it to exist as a monomer. The first coding exon (residues 1 to 72) of wheat germ-expressed Tat was resistant to trypsin digestion, indicating that it is a highly folded, independently structured protein domain. Several mutant Tat proteins were dramatically more sensitive to trypsin than the wild type was, suggesting that their reduced transactivation activities are the result of destabilized structures. Mutant proteins with single-amino-acid substitutions were also identified that had reduced transactivation activities but wild-type structures in the trypsin assay. These mutants clustered in two regions of Tat, at acidic residues 2 and 5 in the amino terminus and between residues 18 and 32. These mutants, wild type in structure but reduced in activity, identify residues in the wild-type protein that may directly contact other molecules during Tat function.