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J D Baxter

Publications and source records attributed to J D Baxter.

At least 37 records · Page 2Linked to original sources
PubMed1999

The human fetal retinal pigment epithelium: A target tissue for thyroid hormones.

Thyroid hormone (T(3)) has previously been shown to regulate visual function in experimental animals and humans. To determine if T(3) exerts direct effects on retinal function, cultured human fetal retinal pigment epithelial (RPE) cells were tested for the presence of thyroid hormone receptors (TRs) and T(3) responses. Using TR-isoform-specific reverse-transcriptase polymerase chain reaction techniques, mRNA was detected for alpha1, alpha2 and beta1 TR isoforms. Immunohistochemistry using a polyclonal antibody that simultaneously recognizes alpha1, alpha2 and beta1 TRs showed nuclear staining of the fetal RPE. Specific binding of (125)I-T(3) to RPE cell nuclear extracts was detected, and Scatchard analysis revealed a K(d) of 110 pM. To determine if RPE cells can respond to T(3), hyaluronic acid (HA) levels in cell culture media were measured after 2, 4 or 6 days of growth in medium containing 10(-7) M T(3). T(3) inhibited accumulation of HA in the cell culture medium of RPE cells. This effect was not evident at 2 days, but at 4 days there was 42.8% less HA in cell culture medium of RPE cells grown in 10(-7) M T(3) (p < 0.01, t test). The effect persisted through 6 days, when there was 46.3% less HA in cell culture medium of RPE cells grown in 10(-7) M T(3) (p < 0.001, t test). The data indicate that human fetal RPE cells are a direct target for thyroid hormones.

Cells, Cultured↗
PubMed1999

Thyroid hormone export in rat FRTL-5 thyroid cells and mouse NIH-3T3 cells is carrier-mediated, verapamil-sensitive, and stereospecific.

Export of L-T3 out of the cell is one factor governing the cellular T3 content and response. We previously observed in liver-derived cells that T3 export was inhibited by verapamil, suggesting that it is due to either ATP-binding cassette/multidrug resistance (MDR1/mdr1b) or multidrug resistance-related (MRP1/mrp1) proteins. To test this hypothesis we measured T3 export in FRTL-5, NIH-3T3, and rat hepatoma (HTC) cells that varied in expression of these proteins. FRTL-5 and NIH-3T3 cells were found to contain a T3 efflux mechanism that is verapamil inhibitable, saturable, and stereospecific. By contrast, T3 efflux in HTC cells was slow and unaffected by verapamil. Neither FRTL-5 nor NIH-3T3 cells express mdrlb, but all three cell types express mrpl, as assessed by immunoblotting. Overexpression of MDR1 in NIH-3T3 cells did not enhance verapamil-inhibitable T3 efflux. Photoaffinity labeling of FRTL-5 and NIH-3T3 cells with [125I]L-T3 revealed a labeled 90- to 100-kDa protein that was not present in HTC cells. Verapamil and excess nonradioactive L-T3, but not D-T3, inhibited labeling of this protein. The lack of correlation between T3 efflux and MDR1 and mrpl expression and the finding of a photoaffinity-labeled putative transport protein smaller than MDR1 or mrp1 protein (approximately 170 kDa) suggest that a novel protein is involved in the transport of T3 out of cells.

3T3 Cells↗
PubMed1999

Titration by estrogen receptor activation function-2 of targets that are downstream from coactivators.

Cross-interference (squelching) among nuclear receptors has been proposed to reflect the titration of coactivators that bind the receptors in a hormone-dependent manner. We have tested whether the coactivators are the only target titrated during squelching of one receptor by another, or whether proteins needed for coactivator function are titrated as well. That the coactivators are indeed one target of squelching is apparent. The isolated ligand-binding domain of the estrogen receptor (ER-LBD) squelches transcriptional activation by the thyroid hormone receptor (TR) only when the LBD is bound to ligands that promote coactivator interactions and only when regions of the LBD that promote coactivator interactions are undisturbed. Furthermore, the ER-LBD and the TR compete in vitro for the related p160 coactivators, SRC1a and GRIP1 (glucocorticoid receptor interacting protein 1), or the putative corepressor, RIP140. Finally TR action becomes more potent when coactivator levels are raised. Nonetheless, supplying excess SRC1a or GRIP1 does not abolish squelching by the ER. In fact, squelching becomes even more severe when coactivators are abundant. Supplying combinations of coactivators from the p160 class and the CREB-binding protein (CBP)/p300 class makes squelching most severe. Elevated RIP140 inhibits TR action, but also protects the residual TR action from squelching by the ER-LBD. We conclude that ER-LBD squelches TR both by titrating p160-CBP coactivators and additionally by cooperating with the coactivators to titrate a second factor. The second factor would be needed by the TR for coactivator-mediated transcriptional stimulation.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Structure and specificity of nuclear receptor-coactivator interactions.

Combinatorial regulation of transcription implies flexible yet precise assembly of multiprotein regulatory complexes in response to signals. Biochemical and crystallographic analyses revealed that hormone binding leads to the formation of a hydrophobic groove within the ligand binding domain (LBD) of the thyroid hormone receptor that interacts with an LxxLL motif-containing alpha-helix from GRIP1, a coactivator. Residues immediately adjacent to the motif modulate the affinity of the interaction; the motif and the adjacent sequences are employed to different extents in binding to different receptors. Such interactions of amphipathic alpha-helices with hydrophobic grooves define protein interfaces in other regulatory complexes as well. We suggest that these common structural elements impart flexibility to combinatorial regulation, whereas side chains at the interface impart specificity.

Amino Acid Sequence↗
PubMed1998

Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors.

The ligand-binding domain of nuclear receptors contains a transcriptional activation function (AF-2) that mediates hormone-dependent binding of coactivator proteins. Scanning surface mutagenesis on the human thyroid hormone receptor was performed to define the site that binds the coactivators, glucocorticoid receptor-interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1). The residues involved encircle a small surface that contains a hydrophobic cleft. Ligand activation of transcription involves formation of this surface by folding the carboxyl-terminal alpha helix against a scaffold of three other helices. These features may represent general ones for nuclear receptors.

HeLa Cells↗
PubMed1998

X-ray crystallographic and functional studies of thyroid hormone receptor.

We have solved several X-ray crystallographic structures of TR ligand-binding domains (LBDs), including the rat (r) TR alpha and the human (h) TR beta bound to diverse ligands. The TR-LBD folding, comprised mostly of alpha-helices, is likely to be general for the superfamily. The ligand, buried in the receptor, forms part of its hydrophobic core. Tight fitting of ligand into the receptor explains its high affinity for the TR, although the structure suggests that ligands with even higher affinities might be generated. The kinetics of 3,5,3'-triiodo-L-thyronine (T3) and 3,5,3',5'-tetraiodo-L-thyronine (T4) binding suggest that folding around the ligand, rather than receptor opening, is rate-limiting for high affinity binding. TR beta mutations in patients with resistance to T3 cluster around the ligand; these different locations could differentially affect on other receptor functions and explain the syndrome's clinical diversity. Guided by the structure, mutations have been placed on the TR surface to define interactions with other proteins. They suggest that a similar surface in the LBD is utilized for homo- or heterodimerization on direct repeats and inverted palindromes but not on palindromes. Coactivator proteins that mediate TR transcriptional activation bind to a small surface comprised of residues on four helices with a well-defined hydrophobic cleft, which may be a target for pharmaceuticals. The coactivator-binding surface appears to form upon ligand-binding by the folding of helix 12 into the scaffold formed by helices 3, 4 and 5. The analysis of most currently used antagonists suggest that although they probably fit into the ligand-binding pocket, they possess a group that may alter proper folding of the receptor, with disruption of the coactivator-binding surface (the 'extension model').

Animals↗
PubMed1998

A high-affinity subtype-selective agonist ligand for the thyroid hormone receptor.

BACKGROUND: Thyroid hormones regulate many different physiological processes in different tissues in vertebrates. Most of the actions of thyroid hormones are mediated by the thyroid hormone receptor (TR), which is a member of the nuclear receptor superfamily of ligand-activated transcription regulators. There are two different genes that encode two different TRs, TR alpha and TR beta, and these two TRs are often co-expressed at different levels in different tissues. Most thyroid hormones do not discriminate between the two TRs and bind both with similar affinities. RESULTS: We have designed and synthesized a thyroid hormone analog that has high affinity for the TRs and is selective in both binding and activation functions for TR beta over TR alpha. The compound, GC-1, was initially designed to solve synthetic problems that limit thyroid hormone analog preparation, and contains several structural changes with respect to the natural hormone 3,5,3'-triiodo-L-thyronine (T3). These changes include replacement of the three iodines with methyl and isopropyl groups, replacement of the biaryl ether linkage with a methylene linkage, and replacement of the amino-acid sidechain with an oxyacetic-acid sidechain. CONCLUSIONS: The results of this study show that GC-1 is a member of a new class of thyromimetic compounds that are more synthetically accessible than traditional thyromimetics and have potentially useful receptor binding and activation properties. The TR beta selectivity of GC-1 is particularly interesting and suggests that GC-1 might be a useful in vivo probe for studying the physiological roles of the different thyroid hormone receptor isoforms.

Animals↗
PubMed1998

DAX1 mutations map to putative structural domains in a deduced three-dimensional model.

The DAX1 protein is an orphan nuclear hormone receptor based on sequence similarity in the putative ligand-binding domain (LBD). DAX1 mutations result in X-linked adrenal hypoplasia congenita (AHC). Our objective was to identify DAX1 mutations in a series of families, to determine the types of mutations resulting in AHC and to locate single-amino-acid changes in a DAX1 structural model. The 14 new mutations identified among our 17 families with AHC brought the total number of families with AHC to 48 and the number of reported mutations to 42; 1 family showed gonadal mosaicism. These mutations included 23 frameshift, 12 nonsense, and six missense mutations and one single-codon deletion. We mapped the seven single-amino-acid changes to a homology model constructed by use of the three-dimensional crystal structures of the thyroid-hormone receptor and retinoid X receptor alpha. All single-amino-acid changes mapped to the C-terminal half of the DAX1 protein, in the conserved hydrophobic core of the putative LBD, and none affected residues expected to interact directly with a ligand. We conclude that most genetic alterations in DAX1 are frameshift or nonsense mutations and speculate that the codon deletion and missense mutations give insight into the structure and function of DAX1.

Adrenal Glands↗
PubMed1998

Molecular and structural biology of thyroid hormone receptors.

1. Thyroid hormone receptors (TR) are expressed from two separate genes (alpha and beta) and belong to the nuclear receptor superfamily, which also contains receptors for steroids, vitamins and prostaglandins. 2. Unliganded TR are bound to DNA thyroid hormone response elements (TRE) predominantly as homodimers, or as heterodimers with retinoid X-receptors (RXR), and are associated with a complex of proteins containing corepressor proteins. Ligand binding promotes corepressor dissociation and binding of a coactivator. 3. Recent studies from our group have focused on the acquisition and use of X-ray crystallographic structures of ligand-binding domains (LBD) of both the rat (r) TR alpha and the human (h) TR beta bound to several different ligands. We have also developed ligands that bind selectively to the TR beta, which may provide ways to explore the differential functions of TR alpha compared with TR beta isoforms. 4. The LBD is comprised mostly of alpha-helices. The ligand is completely buried in the receptor and forms part of its hydrophobic core. Kinetic studies suggest that the limiting step in formation of high-affinity ligand-receptor complexes is the rate of folding of the receptor around the ligand. Ligands can be fitted tightly in the ligand-binding pocket and small differences in this fitting may explain many structure-activity relationships. Interestingly, analysis of the structures of antagonists suggests that they have chemical groups, 'extensions', that could impair receptor folding around them and, thus, prevent the agonist-induced conformation changes in the receptor. 5. The TR structures allowed us to see that the mutations that occur in the syndrome of generalized resistance to thyroid hormone are located in the vicinity of the ligand-binding pocket. 6. X-ray structure of the TR has also been used to guide construction of mutations in the TR surface that block binding of various proteins important for receptor function. Studies with these TR mutants reveal that the interfaces for homo- and heterodimerization map to similar residues in helix 10 and 11 and also allow the definition of the surface for binding of coactivators, which appears to be general for nuclear receptors. Formation of this surface, which involves packing of helix 12 of the TR into a scaffold formed by helices 3 and 5, appears to be the major change in the receptor structure induced by hormone occupancy.

Animals↗
PubMed1998

Mechanisms of thyroid hormone action: insights from X-ray crystallographic and functional studies.

This review summarizes the studies conducted in our laboratory on the mechanisms of thyroid hormone action over the past two decades. We have attempted to place our studies on thyroid hormone receptors (TRs) in perspective with the work conducted by other investigators that established their nuclear localization, DNA-binding properties, DNA response elements, and the role of other proteins involved in TR-mediated regulation of gene transcription. Recently, our crystallographic studies of the TR ligand binding domain (LBD) revealed that the ligand has a structural role in the folding of the receptor's hydrophobic core. The analysis of the structure led to biochemical and genetic studies that have defined the surfaces on the TR LBD required for dimerization and binding of coactivator proteins. Placement of the mutations found in patients with the syndrome of generalized resistance to thyroid hormone on the TR LBD revealed that they were restricted to amino acids in the vicinity of the binding pocket for thyroid hormone. The insights gained from the elucidation of the TR LBD structure will provide the basis for the design of compounds with selective agonistic or antagonistic activities.

Animals↗
PubMed1997

Mutations in the conserved C-terminal sequence in thyroid hormone receptor dissociate hormone-dependent activation from interference with AP-1 activity.

A short C-terminal sequence that is deleted in the v-ErbA oncoprotein and conserved in members of the nuclear receptor superfamily is required for normal biological function of its normal cellular counterpart, the thyroid hormone receptor alpha (T3R alpha). We carried out an extensive mutational analysis of this region based on the crystal structure of the hormone-bound ligand binding domain of T3R alpha. Mutagenesis of Leu398 or Glu401, which are surface exposed according to the crystal structure, completely blocks or significantly impairs T3-dependent transcriptional activation but does not affect or only partially diminishes interference with AP-1 activity. These are the first mutations that clearly dissociate these activities for T3R alpha. Substitution of Leu400, which is also surface exposed, does not affect interference with AP-1 activity and only partially diminishes T3-dependent transactivation. None of the mutations affect ligand-independent transactivation, consistent with previous findings that this activity is mediated by the N-terminal domain of T3R alpha. The loss of ligand-dependent transactivation for some mutants can largely be reversed in the presence of GRIP1, which acts as a strong ligand-dependent coactivator for wild-type T3R alpha. There is excellent correlation between T3-dependent in vitro association of GRIP1 with T3R alpha mutants and their ability to support T3-dependent transcriptional activation. Therefore, GRIP1, previously found to interact with the glucocorticoid, estrogen, and androgen receptors, may also have a role in T3R alpha-mediated ligand-dependent transcriptional activation. When fused to a heterologous DNA binding domain, that of the yeast transactivator GAL4, the conserved C terminus of T3R alpha functions as a strong ligand-independent activator in both mammalian and yeast cells. However, point mutations within this region have drastically different effects on these activities compared to their effect on the full-length T3R alpha. We conclude that the C-terminal conserved region contains a recognition surface for GRIP1 or a similar coactivator that facilitates its interaction with the basal transcriptional apparatus. While important for ligand-dependent transactivation, this interaction surface is not directly involved in transrepression of AP-1 activity.

Amino Acid Sequence↗
PubMed1997

Activities in Pit-1 determine whether receptor interacting protein 140 activates or inhibits Pit-1/nuclear receptor transcriptional synergy.

Pituitary-specific transcription of the evolutionarily related rat (r) GH and PRL genes involves synergistic interactions between Pit-1 and other promoter-binding factors including nuclear receptors. We show that Pit-1/thyroid hormone receptor (TR) and Pit-1/estrogen receptor (ER) synergistic activation of the rGH and rPRL promoters are globally similar. Both synergies depend upon the same activation functions in Pit-1 and also require activation function-2 conserved in TR and ER. The activation function-2 binding protein, RIP140, previously thought to be a nuclear receptor coactivator, strongly inhibits both Pit-1/TR and Pit-1/ER synergy. RIP140 inhibition is profoundly influenced, in a promoter-specific fashion, by a synergism-selective function in Pit-1: deletion of Pit-1 amino acids 72-100 switches RIP140 to an activator of Pit-1/ER and Pit-1/TR synergy at the rPRL promoter but not at the rGH promoter. Pit-1 amino acids 101-125 are required for RIP140 inhibition or activation again only at the rPRL promoter. Therefore, functions within one factor can determine the activity of a coactivator binding to its synergistic partner. This promoter context-specific synergistic interplay between transcription factors and coactivators is likely an essential determinant of cell-specific transcriptional regulation.

Adaptor Proteins, Signal Transducing↗