Functional characterization of two AH receptors in the marine fish, Fundulus heteroclitus: TCDD and DNA binding and tissue-specific expression

Abstract

The aryl hydrocarbon receptor (AHR) is a bHLH-PAS family transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) cause altered gene expression and toxicity. We are examining the AHR signaling pathway in Fundulus heteroclitus, an estuarine fish that is capable of developing heritable dioxin resistance. Unlike mammals, in which a single functional AHR has been reported to date, Fundulus has two distinct AHR genes, FhAHR1 and FhAHR2 (Hahn et al. (1997). Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family. Proc. Natl. Acad. Sci. USA, 94, 13743–13748). We now report the full length sequence and functional characterization of these proteins FhAHR1 (942 amino acids; 104 kDa) and FhAHR2 (905 amino acids; 100 kDa) both contain bHLH and PAS domains that share extensive sequence identity with mammalian AHRs. FhAHR1 is more closely related to mammalian AHRs than is FhAHR2. FhAHR1 and FhAHR2 cDNAs were expressed by in vitro transcription/translation and analyzed for ligand and DNA binding. Velocity sedimentation on sucrose gradients showed that both proteins bind [ 3H]-TCDD specifically and with high affinity. Both AHRs exhibit specific, TCDD-, and ARNT-dependent DNA binding. The FhAHR1 transcript is primarily expressed in brain, heart, and ovary, while the FhAHR2 transcript is equally abundant in many tissues. AHR2 orthologs exist also in several other species of bony and cartilaginous fish, suggesting important functional roles for this protein. (NIEHS ES06272, ES07381, and ES05800.)