The aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) protein are thought to mediate many of the effects of halogenated aromatic hydrocarbons. While many aspects of AHR-mediated signal transduction system have been elucidated in mammalian systems over the past decade there are still numerous questions concerning the mechanism whereby TCDD and related congeners mediate toxic/biological effects and the function of the various proteins that contribute to the pathway. In addition, there is only a modest understanding of the proteins involved in AHR-mediated signal transduction in nonmammalian systems. To begin to gain insight into some of these issues, this laboratory has recently isolated cDNAs from Oncorhynchus mykiss that encode two novel proteins with homology to mammalian ARNT. Importantly, the results show that, (I) multiple ARNT transcripts are generated in Oncorhynchus mykiss; (ii) the multiple transcripts may be produced by alternative RNA splicing, (iii) that the proteins expressed from two messages have opposite functions in AHR-mediated signal transduction and (iv) that the change in function may be related to presence of distinct COOH-terminal domains. There are four interrelated specific aims in this proposal that focus on the analysis of the hypothesis that alternative RNA splicing results in the expression of multiple ARNT proteins that can act positively or negatively on AHR-mediated signal transduction.
Aim 1 is the molecular characterization of the Oncorhynchus mykiss Arnt gene to determine the extent of alternate splicing.
Aim 2 is to determine the mechanism whereby the distinct COOH-terminal domains of Oncorhynchus mykiss ARNT isoforms function in AHR-dependent and independent pathways.
Aim 3 is to develop stable cell lines expressing Oncorhynchus mykiss ARNT isoforms for functional analysis of TCDD-dependent and independent gene expression in vivo.
Aim 4 is to determine the spatial and temporal expression of rtARNT messages and proteins in specific tissues of Oncorhynchus mykiss and during development. The use of Oncorhynchus mykiss will provide a greater understanding of the fundamental significance of ARNT, its evolution and its contribution to TCDD-dependent and independent mechanisms involved in cellular function. The relevance of novel studies of ARNT function are highlighted by recent studies that clearly show ARNT function that is independent of the AHR. Additionally, a detailed study of the function, expression and regulation of ARNT has not been completed in any species.
