Nucleic Acids Research, 2000, Vol. 28, No. 14 2634-2642
© 2000 Oxford University Press
The analysis of chimeric human/rainbow trout estrogen receptors reveals amino acid residues outside of P- and D-boxes important for the transactivation function
Equipe d’Endocrinologie Moléculaire de la Reproduction, UPRES-A CNRS 6026, Université de Rennes I, 35042 Rennes cedex, France
The amino acid sequence of rainbow trout estrogen receptor (rtER) is highly conserved in the C domain but presents few similarities in the A/B and E domains with human estrogen receptor 
(hER) [NR3A1]. A previous study has shown that rtER and hER have differential functional activities in yeast Saccharomyces cerevisiae. To determine the domain(s) responsible for these differences, chimeric human/rainbow trout estrogen receptors were constructed. The A/B, C/D or E/F regions of rtER were replaced by corresponding regions of hER and expressed in yeast cells. Ligand-binding and transcription activation abilities of these hybrid receptors were compared with those of wild-type rtER or hER. Surprisingly, our data revealed that the human C/D domains play an important role in the magnitude of transactivation of ER. Two other chimeric ERs carrying either a C or D domain of hER showed that the C domain was responsible for this effect whereas the D domain did not affect hybrid receptor activities. Moreover, a chimeric hER carrying the C domain of rtER showed maximal transcriptional activity similar to that observed with rtER. Gel shift assays showed that, whereas rtER and hER present a similar binding affinity to an estrogen response element (ERE) element, the rtER C domain is responsible for a weaker DNA binding stability compared to those of hER. In addition, the human C domain allows approximately 2 times faster association of ER to an ERE. Utilization of reporter genes containing one or three EREs confirms that rtER requires protein–protein interactions for its stabilization on DNA and that the C domain is involved in this stabilization. Moreover, AF-1 may be implicated in this synergistic effect of EREs. Interestingly, although E domains of these two receptors are much less conserved, replacement of this domain in rtER by its human counterpart resulted in higher estradiol sensitivity but no increase in the magnitude of transactivation. Data from the chimeric receptors, rtER(hC) and hER(rtC), demonstrated that rtER AF-1 and AF-2 activation domains activated transcription in the presence of estradiol similar to both AF-1 and AF-2 hER. This implies that these domains, which show poor sequence homology, may interact with similar basal transcription factors.
* To whom correspondence should be addressed. Tel: +33 2 99 28 29 70; Fax: +33 2 99 28 67 94; Email: farzad.pakdel@univ-rennes1.fr Present address: Fabrice G. Petit, Department of Molecular and Cellular Biology, Baylor College of Medicine, Texas Medical Center, One Baylor Plaza, Houston, TX 77030, USA
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  P. Y. Kunz, H. F. Galicia, and K. Fent Comparison of In Vitro and In Vivo Estrogenic Activity of UV Filters in Fish Toxicol. Sci., April 1, 2006; 90(2): 349 - 361. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.L. Filby and C.R. Tyler Molecular Characterization of Estrogen Receptors 1, 2a, and 2b and Their Tissue and Ontogenic Expression Profiles in Fathead Minnow (Pimephales promelas) Biol Reprod, October 1, 2005; 73(4): 648 - 662. [Abstract] [Full Text] [PDF]
|
|