Analysis of rainbow trout Ah receptor protein isoforms in cell culture reveals conservation of function in Ah receptor-mediated signal transduction.
Two distinct aryl hydrocarbon receptor (AHR) cDNAs have been isolated from rainbow trout. The encoded receptor protein products termed rtAHR2alpha and rtAHR2ss are 97% identical at the amino acid level but are reported to have distinct functions with regard to AHR-mediated gene regulation. To test this hypothesis, the two proteins were evaluated functionally both in vitro and in a Chinese hamster lung cell line, E36. To facilitate analysis, both rtAHR2 isoforms were tagged with the FLAG peptide and could be expressed and quantified in a rabbit reticulocyte lysate. However, both proteins failed to form functional complexes with mammalian or rainbow trout AHR nuclear translocator protein (ARNT) that could associate with xenobiotic response elements (XREs) in a ligand-dependent manner in vitro. In contrast, both proteins exhibited positive function on AHR-mediated signaling when expressed in the E36 cell line. Both rtAHR2 isoforms showed a cytoplasmic distribution in the unliganded state and could drive the expression of a reporter gene under control of the trout CYP1A3 promoter. Although both proteins induced reporter gene activity to the same magnitude, the EC(50) values of the two isoforms for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) differed by an order of magnitude, with the rtAHR2ss isoform less responsive to TCDD. When the functions of the rtAHR2 isoforms were tested in the context of the dominant negative rtARNT(a) protein, TCDD-mediated induction of reporter gene activity was reduced as the level of rtARNT(a) protein increased. In summary, both rtAHR2 isoforms appear to exhibit positive function in AHR-mediated signaling, suggesting conservation of function.
Pollenz
Richard S
RS
Department of Biology, University of South Florida, 4202 East Fowler Avenue, SCA 110, Tampa, FL 33620-5200, USA.
Necela
Brian
B
Marks-Sojka
Katherine
K
D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D018929:Cell Culture Techniques; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D017686:Oncorhynchus mykiss; D020033:Protein Isoforms / Q000032:analysis; D018336:Receptors, Aryl Hydrocarbon / Q000032:analysis*; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D020033:Protein Isoforms; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator