Vincent C. Henrich

Arthropod nuclear receptors and their role in molting

The molting process in arthropods is regulated by steroid hormones acting via nuclear receptor proteins. The most common molting hormone is the ecdysteroid, 20‐hydroxyecdysone. The receptors of 20‐hydroxyecdysone have also been identified in many arthropod species, and the amino acid sequences determined. The functional molting hormone receptors consist of two members of the nuclear receptor superfamily, namely the ecdysone receptor and the ultraspiracle, although the ecdysone receptor may be functional, in some instances, without the ultraspiracle. Generally, the ecdysone receptor/ultraspiracle heterodimer binds to a number of ecdysone response elements, sequence motifs that reside in the promoter of various ecdysteroid‐responsive genes. In the ensuing transcriptional induction, the ecdysone receptor/ultraspiracle complex binds to 20‐hydroxyecdysone or to a cognate ligand that, in turn, leads to the release of a corepressor and the recruitment of coactivators. 3D structures of the ligand‐binding domains of the ecdysone receptor and the ultraspiracle have been solved for a few insect species. Ecdysone agonists bind to ecdysone receptors specifically, and ligand–ecdysone receptor binding is enhanced in the presence of the ultraspiracle in insects. The basic mode of ecdysteroid receptor action is highly conserved, but substantial functional differences exist among the receptors of individual species. Even though the transcriptional effects are apparently similar for ecdysteroids and nonsteroidal compounds such as diacylhydrazines, the binding shapes are different between them. The compounds having the strongest binding affinity to receptors ordinarily have strong molting hormone activity. The ability of the ecdysone receptor/ultraspiracle complex to manifest the effects of small lipophilic agonists has led to their use as gene switches for medical and agricultural applications.

The Ecdysteroid Receptor

Publisher Summary This chapter examines the information gathered about the ecdysteroid receptor at three levels and provides brief descriptions and references for the tools used to undertake those experiments. The receptor is viewed as a structural entity with particular emphasis on the sequence characteristics of EcR and USP as well as their biophysical and biochemical properties. These studies build upon the first three properties noted for EcR in its original characterization. The transcriptional function of the ecdysteroid receptor is examined, with particular regard to its interactions with ligand, other protein factors, and promoter elements. This chapter describes a few of the ecdysteroid-inducible systems that have been developed for various applications. The ecdysteroid receptor is viewed from a cellular and developmental perspective in vivo with special emphasis on the spatial and temporal diversity of ecdysteroid-mediated action, along with the approaches used to address these questions. Further, a prognosis is offered concerning unresolved and arising issues surrounding ecdysteroid action via its receptors along with possible experimental technologies and strategies that might clarify them.

Prevalence of noise-induced hearing loss in student musicians

Abstract This study describes the prevalence and characteristics of noise-induced hearing loss (NIHL) in student musicians (N = 329) aged 18–25 years. Students completed a questionnaire regarding exposures before a hearing assessment. NIHL was defined by the presence of a notch 15 dB in depth at 4000 or 6000 Hz relative to the best preceding threshold. Overall prevalence of NIHL was 45%, with 78% of notches occurring at 6000 Hz. The proportion of the total population with bilateral notching at any frequency was 11.5%, mostly occurring at 6000 Hz. There was a significant increase in the frequency of notching in students who reported more than two hours per day of personal practice. There were no significant associations for instrument group or other noise exposures. The data suggest that susceptibility to NIHL among students of music is not uniform and cannot be ascribed solely to the instrument played and other exposures. Students with bilateral losses tend to have deeper notches and may represent a group that has an inherent predisposition to NIHL. Sumario Este estudio describe la prevalencia y las características de la hipoacusia inducida por ruido (NIHL) en estudiantes de música (N = 329) con edades entre 18 y 25 años. Los estudiantes completaron un cuestionario sobre exposición a ruido antes de la evaluación auditiva. Se definió NIHL como la presencia de una muesca de 15dB en 4000 o 6000Hz con relación al mejor umbral precedente. La prevalencia general de NIHL fue de 44%, con 78% de las muescas en 6,000 Hz. La proporción de la población total con muescas bilaterales en cualquier frecuencia fue de 11.5%, en su mayoría a 6,000 Hz. Hubo un incremento significativo en la frecuencia de la muescas en los estudiantes que reportaban más de dos horas al día de práctica profesional. No hubo una asociación significativa con grupos de instrumentos u otra exposición a ruido. Los datos sugieren que la susceptibilidad a NIHL entre los estudiantes de música no es uniforme y no puede atribuirse solamente al instrumento tocado o a otras exposiciones. Los estudiantes con pérdida bilateral tienden a tener muescas más profundas y pueden representar un grupo que tenga una predisposición inherente a la NIHL.

A Novel Ecdysone Receptor Mediates Steroid-Regulated Developmental Events during the Mid-Third Instar of Drosophila

The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E) is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E—signaling through a nuclear receptor heterodimer consisting of EcR and USP—induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC) through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component.

Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster

Abstract Mutants created by site-directed mutagenesis were used to elucidate the function of amino acids involved in ligand binding to ecdysteroid receptor (EcR) and heterodimer formation with ultraspiracle (USP). The results demonstrate the importance of the C-terminal part of the D-domain and helix 12 of EcR for hormone binding. Some amino acids are involved either in ligand binding to EcR (E476, M504, D572, I617, N626) or ligand-dependent heterodimerization as determined by gel mobility shift assays (A612, L615, T619), while others are involved in both functions (K497, E648). Some amino acids are suboptimal for ligand binding (L615, T619), but mediate liganddependent dimerization. We conclude that the enhanced regulatory potential by liganddependent modulation of dimerization in the wild type is achieved at the expense of optimal ligand binding. Mutation of amino acids (K497, E648) involved in the salt bridge between helix 4 and 12 impair ligand binding to EcR more severely than hormone binding to the heterodimer, indicating that to some extent heterodimerization compensates for the deleterious effect of certain mutations. Different effects of the same point mutations on ligand binding to EcR and EcR/USP (R511, A612, L615, I617, T619, N626) indicate that the ligandbinding pocket is modified by heterodimerization

The Ecdysone Receptor Puzzle

The present article reviews some recent findings on the functional ecdysone† receptor which is a heterodimer of two proteins: ecdysone receptor (EcR) and Ultraspiracle (USP). Emphasis is given to some unique aspects of this receptor, in particular to its dimerization, binding to DNA, and transactivation capabilities. The effects of ligands (ecdysone, juvenile hormone) on these functions are discussed. In addition, perspectives on future work on this receptor are outlined, which are shaped by recent progress in the nuclear receptor field in general. This preview part of the present article concerns mainly the 3-D structure of receptor domains, the formation of large supramolecular receptor complexes, their influence on chromatin remodelling, receptor phosphorylation, as well as inter- and intramolecular cross-talks of receptor domains.

Educational Needs of Primary Care Physicians Regarding Direct-to-Consumer Genetic Testing

To assess the educational needs of North Carolina primary care physicians (PCPs) about direct-to-consumer (DTC) genetic testing, surveys were mailed to 2,402 family and internal medicine providers in North Carolina. Out of 382 respondents, 323 (85%) felt unprepared to answer patient questions and 282 (74%) reported wanting to learn about DTC genetic testing. A total of 148 (39%) were aware of DTC genetic testing. Among these, 63 (43%) thought DTC genetic testing was clinically useful. PCPs who felt either unprepared to answer patient questions (OR = 0.354, p = 0.01) or that DTC genetic testing was clinically useful (OR = 5.783, p = 0.00) were more likely to want to learn about DTC genetic testing. PCPs are interested in learning about DTC genetic testing, but are mostly unaware of DTC testing and feel unprepared to help patients with DTC testing results. Familiar and trusted channels that provide the information and tools PCPs need to help answer patient’s questions and manage their care should be used when creating educational programs.

Analysis of transcriptional activity mediated by Drosophila melanogaster ecdysone receptor isoforms in a heterologous cell culture system

Ecdysteroid regulation of gene transcription in Drosophila melanogaster and other insects is mediated by a heterodimer comprised of Ultraspiracle (USP) and one of three ecdysone receptor (EcR) isoforms (A, B1 and B2). This study revealed that the EcR/USP heterodimer displays isoform‐specific capabilities. EcRB1 is normally induced with a form of USP that is missing its DNA‐binding domain (DBD), although potentiation by juvenile hormone (JH) III is reduced. The EcRA and B2 isoforms, however, display almost no response to ecdysteroids with the DBD− USP. A mutation, K497E, in the shared ligand‐binding domain of the EcR isoforms caused elevated EcRB2‐specific affinity for a canonical ecdysone response element. The effects of directed modification and mutagenesis offer a strategy for developing hypotheses and considerations for studying in vivo function.

Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster

Abstract The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acidbinding residue within the ligand-binding pocket, I323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.

Developmental Effects of a Chimeric ultraspiracle Gene Derived From Drosophila and Chironomus

Summary: The ultraspiracle (usp) gene encodes a nuclear receptor that forms a heterodimer with the ecdysone receptor (EcR) to mediate transcriptional responses to the insect steroid hormone, 20‐hydroxyecdysone (20HE). The responses ultimately elicit changes associated with molting and metamorphosis. Although Ultraspiracle (USP) is required at several developmental times, it is unclear whether USP plays stage‐specific roles in Drosophila. A chimeric transgene (d/cusp), produced by replacing the ligand‐binding domain (LBD) of Drosophila USP with the equivalent domain from another Diptera, Chironomus tentans, was tested for its ability to rescue Drosophila usp mutants from early larval lethality. A single copy of the d/cusp was sufficient to rescue transformants from several lines through larval development but they died suddenly during the late third instar. Additional doses of d/cusp were required to allow survival through the adult stage, but they did not restore a normal prepupal contraction. Thus, the arrest at the onset of metamorphosis apparently is caused by the impaired ability of the chimeric USP to mediate a stage‐specific function associated with the LBD. genesis 28:125–133, 2000.