conformation change ͉ FQNLF ͉ FRET ͉ nuclear receptor ͉ estrogen receptor T he nuclear receptor (NR) superfamily consists of a large group of ligand-regulated transcription factors. Several NRs are implicated in human physiology and disease (1, 2) and activation of the estrogen receptors (ER) and androgen receptors (AR) are predisposing factors for breast (3) and prostate cancer (4). Indeed, pharmacologic antagonists of AR and ER are used as antineoplastic agents in these diseases (4-7). It is commonly believed that understanding NR structure and function will facilitate development of specific drugs that can replace or supplement current therapies (2). Ligand binding alters NR structure, cofactor interactions, and transcriptional activity (8). Transcriptional activation functions are present in the aminoterminal domain (NTD; AF-1) and the ligand binding domain (LBD; AF-2) of many NRs, including AR (9) and ER (10). AF-1 is not conserved at the primary sequence level and is poorly characterized functionally (11). In contrast, AF-2 is highly conserved (12) and consists of amino acids that form a coactivator binding pocket on the surface of most NR LBDs (13-16).In many NRs, both AF-1 and AF-2 activities are suppressed in the absence of ligand and enabled after ligand binding (9, 10), which implies that ligand binding to the LBD somehow unmasks AF-1 activities in the NTD. The molecular͞structural basis for LBD communication with AF-1 in full-length molecules remains uncertain. However, an intermolecular interaction between NTD peptides and the agonist-bound LBD has been extensively characterized in vitro and with intracellular two-hybrid assays for the AR (14, 17-21) and ER (22). In the AR NTD, deletion or mutation of a sequence ( 23 FQNLF 27 ) that can bind the AF-2 coactivator pocket of the LBD (14, 19) diminishes activity of the AR at certain promoter elements (21). This finding suggests that an NTD-LBD interaction is functionally important, but it remains unknown whether the NTD interacts with the LBD within one molecule or whether it participates in an intermolecular interaction with the LBD of a second AR molecule.Of the currently available experimental approaches, FRET (23) uniquely can resolve conformation changes and protein interactions of the intact NR molecule in living cells. FRET allows real-time detection of protein conformation changes based on energy transfer between fluorophores attached to domains of interest. Here, we used FRET to determine the time and subcellular location of ligand-induced conformational changes in AR that underlie its activity as a transcription factor. We contrasted these studies with other members of the NR family, ER␣ and peroxisome proliferator-activated receptor-␥2 (PPAR␥2), and have determined a role for the AR-specific 23 FQNLF 27 motif in coordinating intramolecular AR conformational changes that precede AR self-association, most likely as a dimer. Materials and MethodsPlasmid Construction. Plasmids that express AR, ER␣, or PPAR␥2 as enhanced cyan f luorescent protein ...

Selective therapeutics for nuclear receptors would revolutionize treatment for endocrine disease. Specific control of nuclear receptor activity is challenging because the internal cavities that bind hormones can be virtually identical. Only one highly selective hormone analog is known for the thyroid receptor, GC-24, an agonist for human thyroid hormone receptor ␤. The compound differs from natural hormone in benzyl, substituting for an iodine atom in the 3 position. The benzyl is too large to fit into the enclosed pocket of the receptor. The crystal structure of human thyroid hormone receptor ␤ at 2.8-Å resolution with GC-24 bound explains its agonist activity and unique isoform specificity. The benzyl of GC-24 is accommodated through shifts of 3-4 Å in two helices. These helices are required for binding hormone and positioning the critical helix 12 at the C terminus. Despite these changes, the complex associates with coactivator as tightly as human thyroid hormone receptor bound to thyroid hormone and is fully active. Our data suggest that increased specificity of ligand recognition derives from creating a new hydrophobic cluster with ligand and protein components.A ll metazoan life depends on transcription control by the family of nuclear receptors. Nuclear receptors regulate development and differentiation as well as metabolism and physiology, and their dysfunction contributes to disorders such as diabetes, obesity, cardiovascular disease, and cancer (1). Synthetic hormone analogs have therapeutic potential for altering the function of many nuclear receptors, provided that they are receptor and isoform selective. Agonist ligands of peroxisome proliferator-activated receptor ␥ are currently used to treat type II diabetes (2-4). Estrogen analogs called selective estrogen receptor modulators that selectively block or activate estrogen receptor isoforms are applied in the therapy of breast cancer and osteoporosis (5, 6).Although investigations on structure-function relationships show that nuclear receptors possess unique features in regulation, their three-dimensional structures are similar. The ligandbinding domain (LBD) binds hormone and is interdependent on other domains that bind to DNA and coregulators or respond to posttranslational modifications (7). Within the LBD, the critically placed C-terminal helix 12 changes its position and binding surface in an allosteric response to hormone binding (8). The function of this conformational change is to shape the surface for binding of coregulators (9, 10). The coactivator complex attracts further cofactors, which are required for activation of the transcription of target genes (11, 12). The shape and size of the hormone-binding pocket, usually completely buried inside the protein, place severe restrictions on the design of ligands. Any subtle changes in the chemical structure of the hormone might alter the position of helix 12 and so determine the fate of the receptor as repressed or activated.The synthesis and evaluation of ligands for thyroid hormone receptor (...

No abstract

AMPA receptors mediate fast excitatory synaptic transmission and are essential for synaptic plasticity. ANQX, a photoreactive AMPA receptor antagonist, is an important biological probe used to irreversibly inactivate AMPA receptors. Here, using X-ray crystallography and mass spectroscopy, we report that ANQX forms two major products in the presence of the GluR2 AMPAR ligand-binding core (S1S2J). Upon photostimulation, ANQX reacts intramolecularly to form FQX or intermolecularly to form a covalent adduct with Glu705.