Reciprocal mutagenesis between human alpha(L349, M528) and rainbow trout (M317, I496) estrogen receptor residues demonstrates their importance in ligand binding and gene expression at different temperatures.

Several fish proteins exhibit compromised function at temperatures outside of their normal physiological range. In this study, the effect of temperature on the ligand binding and the transactivation abilities of the rainbow trout estrogen receptor (rtER) and human estrogen receptor alpha (hER alpha) were examined. Saturation analysis and gene expression assays, using GST-ER and Gal4-ER fusion proteins consisting of the D, E and F domains of human (hER alpha def) and rainbow trout (rtERdef) receptors, show that GST-rtERdef E2 binding affinity and transactivation ability decrease with increasing temperature. A comparison of the amino acid sequence differences between their ligand binding pockets identified two conservative amino acid residue substitutions in rtER (M317, I496) and hER alpha (L349, M528). The effect of these substitutions on ligand binding and transactivation were examined by constructing reciprocal mutants, which effectively exchanged the binding pockets between rtER and hER alpha. The rtERdef M317L:I496M double mutant exhibited increased E2 binding affinity and transactivation ability at higher temperatures, and displayed hER alpha phenotypic behavior for the phytoestrogen, coumestrol. The hER alpha def L349M:M528I double mutant also exhibited a modest trend towards adopting the rtER phenotype. These studies demonstrate that conservative changes in residue hydrophobicity and volume can significantly affect ER ligand binding and transactivation ability in a temperature-dependent manner. The lack of a complete exchange of phenotypes between rtER and hER alpha indicates that factors outside of the ligand binding pocket are also involved.