Temperature dependence of estrogen binding: importance of a subzone in theligand binding domain of a novel piscine estrogen receptor

The full length estrogen receptor from Oreochromis aureus (OaER) was cloned and expressed in vitro and in vivo as a functional transcription factor. A mino acid residues involved in the thermal stability of the receptor are lo cated at/near subzones beta 1 and beta 3, which are highly conserved in oth er non-piscine species but not in OaER. Hormone binding studies, however, i ndicate that OaER is thermally stable but exhibited a similar to 3-fold red uced affinity for estrogen at elevated temperatures. Transfection of OaER i nto various cell lines cultured at different temperatures displayed a signi ficant estrogen dose-response shift compared with that of chicken ER (cER). At 37 degrees C, OaER requires similar to 80-fold more estrogen to achieve half-maximal stimulation of CAT. Lowering of the incubation temperature fr om 37 degrees C to 25 degrees C or 20 degrees C resulted in a 4-fold increa se in its affinity for estrogen. The thermally deficient transactivation of OaER at temperatures above 25 degrees C was fully prevented by high levels of estrogen. Thus, compared to cER, the OaER exhibits reduced affinity for estrogen at elevated temperature as reflected in its deficient transactiva tion capability. Amino acid replacements of OaER beta 3 subzones with corre sponding amino acids from cER could partially rescue this temperature sensi tivity. The three-dimensional structure of the OaER ligand binding domain ( LBD) was modelled based on conformational similarity and sequence homology with human RXR alpha ape, RAR gamma hole and ER alpha LBDs. Unliganded and 17 beta-estradiol-liganded OaER LED retained the overall folding pattern of the nuclear receptor LBDs. The residues at/near the subzone beta 3 of the LED constitute the central core of OaER structure. Thus, amino acid alterat ion at this region potentially alters the structure and consequently its te mperature-dependent ligand binding properties. (C) 1999 Elsevier Science B. V. All rights reserved.