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CLONING, EXPRESSION AND CHARACTERIZATION OF THE ANDROGEN RECEPTOR AND ISOLATION OF ESTROGEN RECEPTOR ALPHA FROM THE FATHEAD MINNOW (PIMEPHALES PROMELAS)
Wilson, V S., M C. Cardon, J. Thornton, J J. Korte, J. E. WELCH, L. E. GRAY, JR., AND P. C. HARTIG. CLONING, EXPRESSION AND CHARACTERIZATION OF THE ANDROGEN RECEPTOR AND ISOLATION OF ESTROGEN RECEPTOR ALPHA FROM THE FATHEAD MINNOW (PIMEPHALES PROMELAS). ENVIRONMENTAL SCIENCE AND TECHNOLOGY 38(23):6314-21, (2004).
This work details the preparation of a cDNA library from fathead minnow
In vitro screening assays designed to identify hormone mimics or antagonists, including those recommended for use in the EPA's Tier 1 screening battery, typically use mammalian estrogen (ER) and androgen receptors (AR) such as rat or human. Although we know that the amino acid sequences of steroid receptors of nonmammalian vertebrates are not identical to the mammalian receptors such as rat or human, a great deal of uncertainty exists about whether these differences impact receptor binding to endocrine disrupting chemicals (EDC). This work details the preparation of a cDNA library from fathead minnow, the isolation and sequencing of both an androgen receptor and estrogen receptor alpha followed by expression and characterization of a functional fathead minnow androgen receptor. cDNA sequences encoding the complete open reading frame for each an androgen and estrogen receptor were isolated, encoding 839 and 560 amino acids, respectively. Comparison of the predicted amino acid sequences showed high homology with AR or ER of other species. Phylogenetic analysis indicates that the fathead ER is of the ER alpha type and clusters with the ER alpha of other Cyprinids as expected. Saturation and competitive binding assays with the fathead AR (fhAR) expressed in COS cells indicate that the receptor binds to androgen with high affinity. Saturation experiments determined that the Kd of the fhAR for R1881, a tritiated synthetic androgen, was 1.8 nM which is comparable to that for the human AR in the same assay system. In COS whole cell competitive binding assays, potent androgens such as DHT and 11-ketotestosterone were shown to be high affinity ligands for the fhAR. Future plans include comparison of binding affinities of the fathead minnow androgen receptor to that of the human androgen receptor using a range of endocrine disrupting chemicals. This will allow comparison of binding affinities of the two receptors within the same system.