Structural biology of arsenic-glucocorticoid receptor interaction.

Lauren Kingsley
Dartmouth College

Arsenic (As) is an environmental human health concern in the US and worldwide. Human epidemiological studies have shown that exposure to elevated levels of naturally occurring arsenic in drinking water are strongly associated with a increased risk of vascular and cardiovascular disease, diabetes, reproductive and developmental effects, and over a dozen different cancers. Arsenic levels at or near the current US EPA current drinking water standard are associated with substantially increased risk of these diseases, and such levels can be found in many areas of the US affecting over 25 million people. Previous studies in our laboratory have shown that arsenic has the ability to act as an endocrine disruptor, binding at very low levels to the glucocorticoid receptor (GR) and almost completely blocking hormone-mediated activation of gene expression by this receptor in cultured cells and in a whole animal model. At the cell physiological level, As was shown to block GR hormone-mediated control of H4IIE cell growth. Previous studies of N-terminal and ligand binding domain (LBD) deletion GR mutants in a GR deficient H4IIE-derived cell line showed that effects of As on GR signaling most closely mapped to the central DNA-binding domain (DBD) of the receptor. Mutation of single amino acids within the DBD altered levels of gene expression in cultured cells in response to As, suggesting that this region is the primary target for As action. More recently, studies in our laboratory have found species differences in the response of human, mouse and rat GR to varying levels of As. As had little or no effect on basal expression of these constructs in the absence of hormone. The lowest doses of As used (0.01 - 0.5 uM) potentiated the hormone-induced gene expression by 2- to 3-fold over that with hormone alone in the rat and mouse. In contrast, slightly higher doses of As (1-3 uM) caused a dose-dependent suppression of induction. Less potentiation of gene expression at low As doses was observed for human GR; however, similar levels of suppression were observed at higher As doses. Rat GR was intermediate in As response in comparison to human and mouse GR. The DBD is highly conserved among the human, mouse and rat, with only three amino acid differences. It is hypothesized that As affects the three-dimensional structure of GR and its ability to interact with other proteins important for transcription. Amino acid changes within the DBD may affect As-GR interaction and lead to the differences in gene expression response observed at low As doses for these species. Site-directed mutagenesis will be performed to convert the DBD of human GR into rat and mouse forms. Data will be presented that shows the effects of mutagenesis on response of human, rat and mouse GR to As treatment. These data should provide insight into the structural basis for the potent effects of As on steroid hormone-mediated gene expression which may play a role in As-induced human disease.