Temperature dependence of estrogen binding: importance of a subzone in the ligand 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. Amino acid residues involved in the thermal stability of the receptor are located at/near subzones beta1 and beta3, which are highly conserved in other non-piscine species but not in OaER. Hormone binding studies, however, indicate that OaER is thermally stable but exhibited a approximately 3-fold reduced affinity for estrogen at elevated temperatures. Transfection of OaER into various cell lines cultured at different temperatures displayed a significant estrogen dose-response shift compared with that of chicken ER (cER). At 37 degrees C, OaER requires approximately 80-fold more estrogen to achieve half-maximal stimulation of CAT. Lowering of the incubation temperature from 37 degrees C to 25 degrees C or 20 degrees C resulted in a 4-fold increase 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 transactivation capability. Amino acid replacements of OaER beta3 subzones with corresponding amino acids from cER could partially rescue this temperature sensitivity. The three-dimensional structure of the OaER ligand binding domain (LBD) was modelled based on conformational similarity and sequence homology with human RXRalpha apo, RARgamma holo and ERalpha LBDs. Unliganded and 17beta-estradiol-liganded OaER LBD retained the overall folding pattern of the nuclear receptor LBDs. The residues at/near the subzone beta3 of the LBD constitute the central core of OaER structure. Thus, amino acid alteration at this region potentially alters the structure and consequently its temperature-dependent ligand binding properties.
Tan
N S
NS
National University of Singapore, Marine Biotechnology Laboratory, Department of Biological Sciences, 10 Kent Ridge Crescent, Singapore, Singapore.
Frecer
V
V
Lam
T J
TJ
Ding
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D004967:Estrogens / Q000737:chemistry / Q000378:metabolism*; D015870:Gene Expression; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011960:Receptors, Estrogen / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature; D017210:Tilapia / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D018076:DNA, Complementary; D004967:Estrogens; D008024:Ligands; D011960:Receptors, Estrogen