Understanding the molecular details of dioxin/Ah receptor (AHR) signal transduction will yield a number of benefits. First, identifying modifiers of AHR signaling will help explain the tissue and species specificity of dioxin toxicity and thus will aid attempts to predict the human and environmental risk that results from exposure to these pollutants. Second, the AHR is a prototype of a large super family of environmental sensors. What we learn here will shed significant light on a variety of environment-gene interactions. Third, it is likely that the AHR will be involved in a variety of human disorders. Detailed information about signaling can guide the pharmacology necessary to develop related therapeutics. Finally, identifying endogenous activators of the AHR will provide one of the most significant clues as to how and why this protein signals in development and begin to explain the conservation of this protein throughout evolution. In an effort to answer these questions, we propose the following specific aims:
Aim 1 : Identify/understand modifiers of AHR signaling through the use of genetic screens in the yeast, S cerevisiae. We will employ two screening approaches in yeast. First we will use a library of deletions to determine the role of each yeast gene product on AHR signal transduction. Second, we will expand upon these screens by using mammalian cDNAs to perform high copy modifier screens in yeast. Identified modifiers will then be characterized using both statistical clustering of their pharmacological action, as well as biochemical approaches to elucidate mechanism.
Aim 2 : Understand the mechanism of AHR modifiers through the use of signaling kinetics in mammalian cell culture. We propose to use fluorescently tagged molecules and reporters to elucidate the cellular mechanisms of AHR modifiers using a mammalian culture system. Once developed, this system will also serve as the basis for an activator screen described in Aim 3.
Aim 3 : Determine how the AHR signals during development. The phenotype of Ah null mice suggests the existence of a developmental activator of the AHR. To identify this factor, we propose to: 1) identify those developmental sites where the activator is most likely to be expressed;2) screen for cDNAs encoding protein factors that activate the AHR using an activator trap protocol in cell culture;and 3) characterize the soluble factor derived from murine heart that activates the AHR in cell culture.
Aim 4 : Complete the high-resolution domain mapping studies of the AHR.
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