PHOTOAFFINITY-LABELING OF THE AH RECEPTOR - PHYLOGENETIC SURVEY OF DIVERSE VERTEBRATE AND INVERTEBRATE SPECIES

The mammalian aromatic hydrocarbon (Ah) receptor is a soluble protein involved in the regulation of gene expression by halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Littl e is known, however, about the presence and properties of this recepto r in nonmammalian species. In these studies, we sought evidence for an Ah receptor in the liver or liver-equivalent of diverse species of in vertebrate and vertebrate animals. Velocity sedimentation analysis of hepatic cytosol labeled with [H-3]TCDD gave equivocal results with thr ee species of marine fish. In subsequent studies, photoaffinity labeli ng with 2-azido-3-[I-125]iodo-7,8-dibromodibenzo-p-dioxin was used to identify the Ah receptor. Specific labeling (labeling that could be di splaced by an excess of unlabeled ligand) was observed in seven specie s of teleost and elasmobranch fish, including winter flounder (Pleuron ectes americanus), killifish (Fundulus heteroclitus), scup (Stenotomus chrysops), rainbow trout (Oncorhynchus mykiss), brown trout (Salmo tr utta), and dogfish (Mustelus canis and Squalus acanthias). Specific la beling was also found in cytosolic fractions prepared from PLHC-1 fish hepatoma cells and livers of a turtle (Chrysemys picta) and a cetacea n, the beluga whale Delphinapterus leucas. The fish Ah receptor was se nsitive to conditions of tissue preparation; inclusion of proteinase i nhibitors in the homogenization buffer stabilized the receptor in some species. There was heterogeneity in the apparent molecular mass of th e largest specifically labeled band in each species; these ranged from 105 to 146 kDa, slightly larger on average than mammalian Ah receptor s (95-130 kDa). In contrast to the results obtained with teleost and e lasmobranch fish, no specifically labeled polypeptides were detectable in cytosol from two agnathan fish species (hagfish Myxine glutinosa a nd sea lamprey Petromyzon marinus), the tunicate Ciona intestinalis, o r any of nine other invertebrate species representing eight classes in four phyla. Overall these results suggest that the Ah receptor evolve d at least 450 million years ago, prior to the divergence of bony and cartilaginous fishes. Although the exact relationship between receptor presence and dioxin responsiveness in these species is uncertain, our data predict that the invertebrate species examined in this study, wh ich appear to lack an Ah receptor protein like that seen in mammals an d fish, may be less sensitive than vertebrates to the effects of envir onmental contaminants that act through this transcriptional regulator. (C) 1994 Academic Press, Inc.