Studying Nuclear Receptor Complexes in the Cellular Environment.

The ligand-regulated structure and biochemistry of nuclear receptor complexes are commonly determined by in vitro studies of isolated receptors, cofactors, and their fragments. However, in the living cell, the complexes that form are governed not just by the relative affinities of isolated cofactors for the receptor but also by the cell-specific sequestration or concentration of subsets of competing or cooperating cofactors, receptors, and other effectors into distinct subcellular domains and/or their temporary diversion into other cellular activities. Most methods developed to understand nuclear receptor function in the cellular environment involve the direct tagging of the nuclear receptor or its cofactors with fluorescent proteins (FPs) and the tracking of those FP-tagged factors by fluorescence microscopy.

Advantages and Limitations of Androgen Receptor-Based Methods for Detecting Anabolic Androgenic Steroid Abuse as Performance Enhancing Drugs.

Testosterone (T) and related androgens are performance enhancing drugs (PEDs) abused by some athletes to gain competitive advantage. To monitor unauthorized androgen abuse, doping control programs use mass spectrometry (MS) to detect androgens, synthetic anabolic-androgenic steroids (AASs) and their metabolites in an athlete's urine. AASs of unknown composition will not be detected by these procedures.

Visualization of positive transcription elongation factor b (P-TEFb) activation in living cells.

Regulation of transcription elongation by positive transcription elongation factor b (P-TEFb) plays a central role in determining the state of cell activation, proliferation, and differentiation. In cells, P-TEFb exists in active and inactive forms. Its release from the inactive 7SK small nuclear ribonucleoprotein complex is a critical step for P-TEFb to activate transcription elongation.

Insulin resistance induced by hyperinsulinemia coincides with a persistent alteration at the insulin receptor tyrosine kinase domain.

Insulin resistance, the diminished response of target tissues to insulin, is associated with the metabolic syndrome and a predisposition towards diabetes in a growing proportion of the worldwide population. Under insulin resistant states, the cellular response of the insulin signaling pathway is diminished and the body typically responds by increasing serum insulin concentrations to maintain insulin signaling. Some evidence indicates that the increased insulin concentration may itself further dampen insulin response.

Maximizing the quantitative accuracy and reproducibility of Förster resonance energy transfer measurement for screening by high throughput widefield microscopy.

Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) provides insights into the proximities and orientations of FPs as surrogates of the biochemical interactions and structures of the factors to which the FPs are genetically fused. As powerful as FRET methods are, technical issues have impeded their broad adoption in the biologic sciences. One hurdle to accurate and reproducible FRET microscopy measurement stems from variable fluorescence backgrounds both within a field and between different fields.

A versatile, bar-coded nuclear marker/reporter for live cell fluorescent and multiplexed high content imaging.

The screening of large numbers of compounds or siRNAs is a mainstay of both academic and pharmaceutical research. Most screens test those interventions against a single biochemical or cellular output whereas recording multiple complementary outputs may be more biologically relevant. High throughput, multi-channel fluorescence microscopy permits multiple outputs to be quantified in specific cellular subcompartments.

FRET analysis of androgen receptor structure and biochemistry in living cells.

The androgen receptor (AR) is the central component of a dynamic conformational and interaction cascade initiated by androgenic hormones. AR function can be modified by cellular inputs not examined in test tube studies of AR action. Thus, there is a need to measure AR conformation and biochemistry directly within the cell where the intracellular locations, levels and availability of the hormone, AR, AR-interacting factors, DNA-binding sites, enzymes that modify those components of AR action, and factors that compete for the formation of functional AR-cofactor complexes may affect AR action.

Structure, affinity, and availability of estrogen receptor complexes in the cellular environment.

An ability to measure the biochemical parameters and structures of protein complexes at defined locations within the cellular environment would improve our understanding of cellular function. We describe widely applicable, calibrated Förster resonance energy transfer methods that quantify structural and biochemical parameters for interaction of the human estrogen receptor alpha-isoform (ER alpha) with the receptor interacting domains (RIDs) of three cofactors (SRC1, SRC2, SRC3) in living cells. The interactions of ER alpha with all three SRC-RIDs, measured throughout the cell nucleus, transitioned from structurally similar, high affinity complexes containing two ER alphas at low free SRC-RID concentrations (<2 nm) to lower affinity complexes with an ER alpha monomer at higher SRC-RID concentrations (approximately 10 nm).

Drug and cell type-specific regulation of genes with different classes of estrogen receptor beta-selective agonists.

Estrogens produce biological effects by interacting with two estrogen receptors, ERalpha and ERbeta. Drugs that selectively target ERalpha or ERbeta might be safer for conditions that have been traditionally treated with non-selective estrogens. Several synthetic and natural ERbeta-selective compounds have been identified.

Dimerization between aequorea fluorescent proteins does not affect interaction between tagged estrogen receptors in living cells.

Forster resonance energy transfer (FRET) detection of protein interaction in living cells is commonly measured following the expression of interacting proteins genetically fused to the cyan (CFP) and yellow (YFP) derivatives of the Aequorea victoria fluorescent protein (FP). These FPs can dimerize at mM concentrations, which may introduce artifacts into the measurement of interaction between proteins that are fused with the FPs. Here, FRET analysis of the interaction between estrogen receptors (alpha isoform, ERalpha) labeled with "wild-type" CFP and YFP is compared with that of ERalpha labeled with "monomeric" A206K mutants of CFP and YFP.

Fluorescent protein tools for studying protein dynamics in living cells: a review.

We have witnessed remarkable advances over the past decade in the application of optical techniques to visualize the genetically encoded fluorescent proteins (FPs) in living systems. The imaging of the FPs inside living cells has become an essential tool for studies of cell biology and physiology. FPs are now available that span the visible spectrum from deep blue to deep red, providing a wide choice of genetically encoded fluorescent markers.

A palmitoylation cycle dynamically regulates partitioning of the GABA-synthesizing enzyme GAD65 between ER-Golgi and post-Golgi membranes.

GAD65, the smaller isoform of the enzyme glutamic acid decarboxylase, synthesizes GABA for fine-tuning of inhibitory neurotransmission. GAD65 is synthesized as a soluble hydrophilic protein but undergoes a hydrophobic post-translational modification and becomes anchored to the cytosolic face of Golgi membranes. A second hydrophobic modification, palmitoylation of Cys30 and Cys45 in GAD65, is not required for the initial membrane anchoring but is crucial for post-Golgi trafficking of the protein to presynaptic clusters.

Functional sequestration of transcription factor activity by repetitive DNA.

Higher eukaryote genomes contain repetitive DNAs, often concentrated in transcriptionally inactive heterochromatin. Although repetitive DNAs are not typically considered as regulatory elements that directly affect transcription, they can contain binding sites for some transcription factors. Here, we demonstrate that binding of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) to the mouse major alpha-satellite repetitive DNA sequesters C/EBPalpha in the transcriptionally inert pericentromeric heterochromatin.

Selective activation of estrogen receptor-beta transcriptional pathways by an herbal extract.

Novel estrogenic therapies are needed that ameliorate menopausal symptoms and have the bone-sparing effects of endogenous estrogens but do not promote breast or uterine cancer. Recent evidence suggests that selective activation of the estrogen receptor (ER)-beta subtype inhibits breast cancer cell proliferation. To establish whether ERbeta-selective ligands represent a viable approach to improve hormone therapy, we investigated whether the estrogenic activities present in an herbal extract, MF101, used to treat hot flashes, are ERbeta selective.

Ligand-selective interdomain conformations of estrogen receptor-alpha.

Selective estrogen receptor modulators (SERMs) inhibit estrogen activation of the estrogen receptor (ER) in some tissues but activate ER in other tissues. These tissue-selective actions suggest that SERMs may be identified with tissue specificities that would improve the safety of breast cancer and hormone replacement therapies. The identification of an improved SERM would be aided by understanding the effects of each SERM on the structure and interactions of ER.

The structural basis of androgen receptor activation: intramolecular and intermolecular amino-carboxy interactions.

Nuclear receptors (NRs) are ligand-regulated transcription factors important in human physiology and disease. In certain NRs, including the androgen receptor (AR), ligand binding to the carboxy-terminal domain (LBD) regulates transcriptional activation functions in the LBD and amino-terminal domain (NTD). The basis for NTD-LBD communication is unknown but may involve NTD-LBD interactions either within a single receptor or between different members of an AR dimer.

Imaging molecular interactions in living cells.

Hormones integrate the activities of their target cells through receptor-modulated cascades of protein interactions that ultimately lead to changes in cellular function. Understanding how the cell assembles these signaling protein complexes is critically important to unraveling disease processes, and to the design of therapeutic strategies. Recent advances in live-cell imaging technologies, combined with the use of genetically encoded fluorescent proteins, now allow the assembly of these signaling protein complexes to be tracked within the organized microenvironment of the living cell.

Phosphorylation of C/EBPalpha inhibits granulopoiesis.

CCAAT/enhancer-binding protein alpha (C/EBPalpha) is one of the key transcription factors that mediate lineage specification and differentiation of multipotent myeloid progenitors into mature granulocytes. Although C/EBPalpha is known to induce granulopoiesis while suppressing monocyte differentiation, it is unclear how C/EBPalpha regulates this cell fate choice at the mechanistic level. Here we report that inducers of monocyte differentiation inhibit the alternate cell fate choice, that of granulopoiesis, through inhibition of C/EBPalpha.

Imaging the localized protein interactions between Pit-1 and the CCAAT/enhancer binding protein alpha in the living pituitary cell nucleus.

The homeodomain protein Pit-1 cooperates with the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPalpha) to control pituitary-specific prolactin gene transcription. We previously observed that C/EBPalpha was concentrated in regions of centromeric heterochromatin in pituitary GHFT1-5 cells and that coexpressed Pit-1 redistributed C/EBPalpha to the subnuclear sites occupied by Pit-1. Here, we used fluorescence resonance energy transfer microscopy to show that when C/EBPalpha was recruited by Pit-1, the average distance separating the fluorophores labeling the proteins was less than 7 nm.

A PIT-1 homeodomain mutant blocks the intranuclear recruitment of the CCAAT/enhancer binding protein alpha required for prolactin gene transcription.

The pituitary-specific homeodomain protein Pit-1 cooperates with other transcription factors, including CCAAT/enhancer binding protein alpha (C/EBPalpha), in the regulation of pituitary lactotrope gene transcription. Here, we correlate cooperative activation of prolactin (PRL) gene transcription by Pit-1 and C/EBPalpha with changes in the subnuclear localization of these factors in living pituitary cells. Transiently expressed C/EBPalpha induced PRL gene transcription in pituitary GHFT1-5 cells, whereas the coexpression of Pit-1 and C/EBPalpha in HeLa cells demonstrated their cooperativity at the PRL promoter.

Conformation of CCAAT/enhancer-binding protein alpha dimers varies with intranuclear location in living cells.

The structure of a protein defines its biochemical properties, but the impact of intracellular location and environment on protein structure remains poorly defined. CCAAT/enhancer-binding protein alpha (C/EBPalpha) is a master regulator of transcription and cellular proliferation that concentrates and is kept inactive at transcriptionally quiescent, pericentromeric regions in mouse cell nuclei. C/EBPalpha dimer structure was measured in living cells from the amounts of fluorescence energy transferred between derivatives of the green fluorescent protein attached to different C/EBPalpha domains.

CCAAT/enhancer binding protein alpha uses distinct domains to prolong pituitary cells in the growth 1 and DNA synthesis phases of the cell cycle.

Background: A number of transcription factors coordinate differentiation by simultaneously regulating gene expression and cell proliferation. CCAAT/enhancer binding protein alpha (C/EBPalpha) is a basic/leucine zipper transcription factor that integrates transcription with proliferation to regulate the differentiation of tissues involved in energy balance. In the pituitary, C/EBPalpha regulates the transcription of a key metabolic regulator, growth hormone.