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 ...

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. The intracellular equilibrium dissociation constant for the hormone-induced ERalpha-ERalpha interaction is similar for ERalpha labeled with wild-type or monomeric FPs. However, the measurement of energy transfer measured for ERalpha-ERalpha interaction in each cell is less consistent with the monomeric FPs. Thus, dimerization of the FPs does not affect the kinetics of ERalpha-ERalpha interaction but, when brought close together via ERalpha-ERalpha interaction, FP dimerization modestly improves FRET measurement.

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 För-ster resonance energy transfer methods that quantify structural and biochemical parameters for interaction of the human estrogen receptor ␣-isoform (ER␣) with the receptor interacting domains (RIDs) of three cofactors (SRC1, SRC2, SRC3) in living cells. The interactions of ER␣ with all three SRC-RIDs, measured throughout the cell nucleus, transitioned from structurally similar, high affinity complexes containing two ER␣s at low free SRC-RID concentrations (<2 nM) to lower affinity complexes with an ER␣ monomer at higher SRC-RID concentrations (ϳ10 nM). The methods also showed that only a subpopulation of ER␣ was available to form complexes with the SRC-RIDs in the cell. These methods represent a template for extracting unprecedented details of the biochemistry and structure of any complex that is capable of being measured by Förster resonance energy transfer in the cellular environment.The dynamic processes that are fundamental to life consist of intersecting webs of interactions among biologic factors and cellular signaling pathways (1, 2). Historically, details of interaction kinetics have been measured in test tube studies that compare the level of interaction between purified factors in relationship to the amounts of those purified factors. The extent to which these quantifiers of interaction are modified when challenged by the complex environment and structure of the living cell remains an open question (3-5). For example, an ability to measure the biochemical details of estrogen action within different cellular environments may be necessary to understand tissue-specific estrogen physiology. An ability to perform cellular biochemistry also may improve our knowledge of, and treatments for, the aberrations of estrogen signaling associated with diseases such as breast or uterine cancers (6, 7). This would involve understanding how interactions of the estrogen receptors with any of a large number of cofactors (8 -11) are impacted by cell environment in different tissues of the body.Current techniques that detect interactions between factors in cells include co-immunoprecipitation, two-hybrid analysis, and Förster resonance energy transfer (FRET) 2 microscopy. FRET microscopy senses when factors co-localized in a cellular domain interact in a way that brings fluorophores attached to the factors into close enough proximity to allow energy to transfer from a Donor fluorophore to an Acceptor fluorophore (12-17). All of these methods commonly are interpreted in a simplistic binary "interaction or no interaction" fashion that does not describe quantitative differences among interactions. New technologies are needed to quantify the kinetics of interactions directly in the cell, where the rate and amounts of complexes formed may be affected by the intracellular distributions of the int...

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