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 temperatu re on the ligand binding and the transactivation abilities of the rainbow t rout estrogen receptor (rtER) and human estrogen receptor alpha (hER alpha) were examined. Saturation analysis and gene expression assays, using GST-E R 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-rtERd ef E2 binding affinity and transactivation ability decrease with increasing temperature. A comparison of the amino acid sequence differences between t heir ligand binding pockets identified two conservative amino acid residue substitutions in rtER (M317, 1496) and hER alpha (L349, M528). The effect o f these substitutions on ligand binding and transactivation were examined b y constructing reciprocal mutants, which effectively exchanged the binding pockets between rtER and hER alpha. The rtERdef M317L:1496M double mutant e xhibited increased E2 binding affinity and transactivation ability at highe r temperatures, and displayed hER alpha phenotypic behavior for the phytoes trogen. coumestrol. The hER alpha def L349M:M528I double mutant also exhibi ted a modest trend towards adopting the rtER phenotype. These studies demon strate that conservative changes in residue hydrophobicity and volume can s ignificantly affect ER ligand binding and transactivation ability in a temp erature-dependent manner. The lack of a complete exchange of phenotypes bet ween rtER and hER alpha indicates that factors outside of the ligand bindin g pocket are also involved. (C) 2001 Elsevier Science Ireland Ltd. All righ ts reserved.