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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 ...

A number of conditions, including osteoporosis, frailty, and sexual dysfunction in both men and women have been improved using androgens. However, androgens are not widely used for these indications because of the side effects associated with these drugs. We describe an androgen receptor (AR) ligand that maintains expected anabolic activities with substantially diminished activity in the prostate. LGD2226 is a nonsteroidal, nonaromatizable, highly selective ligand for the AR, exhibiting virtually no affinity for the other intracellular receptors. We determined that AR bound to LGD2226 exhibits a unique pattern of protein-protein interactions compared with testosterone, fluoxymesterone (an orally available steroidal androgen), and other steroids, suggesting that LGD2226 alters the conformation of the ligand-binding domain. We demonstrated that LGD2226 is fully active in cell-based models of bone and muscle. LGD2226 exhibited anabolic activity on muscle and bone with reduced impact on prostate growth in rodent models. Biomechanical testing of bones from animals treated with LGD2226 showed strong enhancement of bone strength above sham levels. LGD2226 was also efficacious in a sex-behavior model in male rats measuring mounts, intromissions, ejaculations, and copulation efficiency. These results with an orally available, nonaromatizable androgen demonstrate the important role of the AR and androgens in mediating a number of beneficial effects in bone, muscle, and sexual function independent from the conversion of androgens into estrogenic ligands. Taken together, these results suggest that orally active, nonsteroidal selective androgen receptor modulators may be useful therapeutics for enhancing muscle, bone, and sexual function.

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