Identification of Residues within Human ERa and Rainbow Trout ER that Contribute to Temperature Sensitivity and Interaction with Estrogenic Compounds. Matthews J.B. and Zacharewski T.R.

The human estrogen receptor a (hERa) and the rainbow trout ER (rtER) have different functional activities in response to E2 and other estrogenic chemicals. Amino acid residues that differ between hERa and rtER ligand binding pockets were identified, L349 and M528 in hERa and M317 and I496 in rtER. Reciprocal mutants, hERa (L349M, M528I) and rtER (M317L, I496M), were prepared. The affinity (Kd value) of GST-ER fusion proteins consisting of the D, E and F domains of hERa (GST-hERa) and rtER (GST-rtER) for E2 was 0.5 nM and 1.0 nM, respectively at 4 C. Although the GST-hERa Kd value was unchanged at 20 C, 30 C and 37 C, that for GST-rtER was 1.8-, 4.4- and 5.2-fold higher at 20 C, 30 C and 37 C, respectively. The Kd value for the M317L;I496M mutant of GST-rtER was 0.85-, 1.5- and 1.8- fold different compared to wild type. The ability of E2 to induce reporter gene activity in transiently transfected MCF-7 cells maintained at 37 C was 280-fold greater for Gal4-hERa than for Gal4-rtER with EC50 values of 0.01 nM and 28 nM, respectively. However, when MCF-7 cells were maintained at 30 C and 20 C only a 30- and 9-fold difference, respectively, was observed. The L349M;M528I mutant of Gal4-hERa caused a 20-fold reduction in E2-induced reporter gene activity at 37 C, which was reduced 3- and 4-fold at 30 C and 20 C respectively. The E2-induced reporter gene activity mediated by the M315L;I496M mutant of Gal4-rtER was reduced 60-fold when compared to that of Gal4-hERa at 37 C. This effect was reduced to 8- and 3-fold at 30 C and 20 C, respectively. In addition, For some chemicals the double mutants adopted the binding preference and ability to activate gene expression of the other wild type receptor. This suggests that residues L349/M317 and M528/I496 play important roles in the observed differences in ligand binding between the hERa and rtER, and in influencing the reduction in E2-induced reporter gene activity and the temperature sensitivity of the rtER.