Abstract
Two aryl hydrocarbon receptors (rtAHR2alpha and rtAHR2beta) have been identified in the rainbow trout (Oncorhynchus mykiss). These receptors share 98% amino acid identity, yet their functional properties differ. Both rtAHR2alpha and rtAHR2beta bind 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dimerize with rainbow trout ARNTb (rtARNTb), and recognize dioxin response elements in vitro. However, in a transient transfection assay the two proteins show differential ability to recognize enhancers, produce transactivation, and respond to TCDD. To identify the sequence differences that confer the functional differences between rtAHR2alpha and rtAHR2beta, we constructed chimeric rtAHRs, in which segments of one receptor form was replaced with the corresponding part from the other isoform. This approach progressively narrowed the region being examined to a single residue, corresponding to position 111 in rtAHR2beta. Altering this residue in rtAHR2beta from the lysine to glutamate found in rtAHR2alpha produced an rtAHR2beta with the properties of rtAHR2alpha. All other known AHRs resemble rtAHR2alpha and carry glutamate at this position, located at the N terminus of the PAS-A domain. We tested the effect of altering this glutamate in the human and zebrafish AHRs to lysine. This lysine substitution produced AHRs with transactivation properties that were similar to rtAHR2beta. These results identify a critical residue in AHR proteins that has an important impact on transactivation, enhancer site recognition, and regulation by ligand.
Highlights
The aryl hydrocarbon receptor (AHR)1 and its associated dimerization partner ARNT are members of the basic helix
TCDD binding causes the AHR protein to dissociate from cytosolic HSP90 and move into the nucleus where it forms a functional dimer with ARNT
Both AHR proteins were able to produce a readily detected reporter signal that was induced by TCDD
Summary
These receptors share 98% amino acid identity, yet their functional properties differ Both rtAHR2␣ and rtAHR2 bind 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD), dimerize with rainbow trout ARNTb (rtARNTb), and recognize dioxin response elements in vitro. PAS domains act as regulated protein interaction surfaces and are involved in a wide variety of sensory/signaling processes in both eukaryotes and prokaryotes These domains are involved in ligand binding to AHR, and the subsequent change in protein associations, subcellular location, and activity. Two AHR genes encoding rtAHR2␣ and rtAHR2 have been identified in rainbow trout These two AHR isoforms are ϳ98% identical in primary sequence. RtAHR2␣ has stronger transactivation properties than rtAHR2 This is somewhat surprising in light of the fact that these two proteins are identical in sequence in the C-terminal domain that is thought to mediate transcriptional activation. These experiments indicate that the functional differences between rtAHR2␣ and rtAHR2 are conferred by a single amino acid difference corresponding to position 111 in rtAHR2
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