Identification of Residues Within Human ERalpha and Rainbow Trout ER that are Important for Interaction with Estrogenic Compounds. J.B. Matthews and T.R. Zacharewski.

The human estrogen receptor alpha (hERalpha) and the rainbow trout ER (rtER) have different functional activities in response to E2 exposure and exhibit different binding preference and affinity for a variety of estrogenic chemicals. Sequence alignment of the hERalpha to the rainbow trout ER (rtER) and existing crystallographic data of the hERa-E2 complex were used to identify amino acid residues that differ between hERa and rtER ligand binding pockets. L349 and M528 in hERalpha and the corresponding residues, M317 and I496 in rtER, were identified and reciprocal mutants, hERalpha (L349M, M528I) and rtER (M317L, I496M), were prepared. The binding affinity of glutathione-S-transferase (GST) fusion proteins consisting of the D, E and F domains of human (alpha), (GST-hERalpha-def) and rainbow trout (GST-rtERdef) ERs was similar among the mutant proteins with Kd values that ranged from 0.5 to 1.0 nM. The ability of E2 to induce reporter gene activity in transiently transfected MCF-7 cells was 280-fold greater for Gal4-hERalpha-def than for Gal4-rtERdef with EC50 values of 0.01 and 28 nM, respectively. The L349M and M528I mutations in hERalpha each caused a 4-fold reduction in the E2 induced response when compared to wild type, while the double mutant caused a 20-fold reduction. The rtER M317L and double mutant resulted in a dose-dependent shift for E2 transactivation by lowering the EC50 value 4.7 fold, while the I496M mutation had no effect on E2 induced reporter gene activity. For some chemicals the double mutants adopted the binding preference of the other wild type receptor. For example, coumestrol exhibited similar affinity to the GST-hERalpha double mutant when compared to the wild type GST-rtER, as would be expected from the reciprocal mutagenesis. In addition, the ability of coumestrol to induce gene expression mediated by the Gal4-hERalpha-def double mutant was reduced 10-fold in comparison to wild type Gal4-hERalpha-def. This suggests that residues L349/M317 and M528/I496 play important roles in the observed differences in ligand binding between the human and rainbow trout ERs and also influence the dose-dependent reduction in E2 induced reporter gene activity.