Affinity labelling and cloning of steroid and thyroid hormone receptors / edited by H. Gronemeyer.

Date:
[1988]
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Licence: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

Credit: Affinity labelling and cloning of steroid and thyroid hormone receptors / edited by H. Gronemeyer. Source: Wellcome Collection.

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    Ch. 15] The nuclear receptor family: cloning, structure and function 277 HEO respectively (data not shown). Experiments with vit-tk-CAT were performed using between 10 ng and 5 ¿ag of the receptor mutant expression vectors, while those with pS2-CAT were performed using 1 and 5 ¡xg. 2. Region С is responsible for the specific recognition of the hormone responsive element Assuming that steroid receptors activate gene transcription by a mechanism which involves the specific recognition of promoter sequences of the target gene, we reasoned that the replacement of the human estrogen receptor DNA-binding domain with that of the human glucocorticoid receptor may produce a chimeric receptor capable of activating glucocorticoid-responsive genes in the presence of estradiol. Since region C, which is responsible for tight nuclear binding, was likely to contain the DNA-binding domain (see above), we decided to prepare such a chimeric receptor by replacing the 66 highly conserved amino acids of region С of the human estrogen receptor by those of the human glucocorticoid receptor (Green and Chambón, 1987; Fig. 14A). To facilitate the exchange of region С between the two receptors, unique restriction enzyme recognition sites were created at the 5'-(KpnI) and 3'-(XhoI) ends of this region in the cDNAs of both the human estrogen receptor and human glucocorticoid receptor (HE28 and GR-CAS; Fig. 14 A). The estrogen receptor cysteine-rich cassette (ER-CAS) was replaced by that of the glucocorticoid receptor (GR-CAS) to produce the novel human estrogen-glucocorticoid receptor chimera ER-GR'CAS (Fig. 14A). Both HE28 and ER-GR-CAS bound estradiol as effec¬ tively as HEO when expressed in HeLa cells (data not shown). These receptor expression vectors, together with HEO, were tested in the presence and absence of estradiol for their ability to activate the estrogen-inducible reporter gene vit-tk-CAT in HeLa cells. In the presence of estradiol, CAT activity was stimulated by both HEO and HE28, whereas ER-GR-CAS and the parent expression vector, pKCR2 (which does not contain receptor sequences) had no effect (Fig. 14B). These receptors were also tested using the glucocorticoid-inducible reporter gene, MMTV-CAT. This gene contains the mouse mammary tumor virus (MMTV) long terminal repeat (-631 to +125) upstream of the CAT gene (Cato et al., 1986). Whereas HEO, HE28, and pKCR2 were not able to stimulate MMTV-CAT, stimulation with ER-GR-CAS was seen only in the presence of estradiol (Fig. 14B), and the concentration of estradiol required for activation was similar to that required for HEO to stimulate vit-tk-CAT (our unpublished results), suggesting that the mechanism by which estradiol acti¬ vates these receptors is similar. Thus region С of steroid receptors determines the specificity of target gene activation by the hormone. The reverse 'finger-swapping' experiment has been performed which gives a GR-ER-CAS chimera which is able to transactivate transcription from the ERE of estrogen receptor target genes under the influence of glucocorticoids (Kumar et al., 1987). A very important application of this technique is the identification of ligands of unknown receptor genes belonging to the same superfamily (see Figs. 8 and 10) as demonstrated in our laboratory by Petkovich et al. (1987). In their case a cDNA was isolated on the basis of crosshybridization with a synthetic oHgonucleotide probe (see also Chapter 14) which contained an open reading frame with the structural characteristics of a 'finger' receptor (Fig. 8). No ligand or target gene of this receptor