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 283 glucocorticoid receptor, which lack all or part of the hormone binding domain, are able to constitutively activate MMTV-CAT (Godowski et al., 1987; Hollenberg et al., 1987). In the case of one human glucocorticoid receptor deletion mutant this constitutive activity is at least as great as that of the occupied wild type receptor (Hollenberg et al., 1987). One possible interpretation of these results is that the unoccupied hormone binding domain exerts some negative influence on the activity of the receptor which is relieved when the hormone binds. The results with insertion mutants of the human glucocorticoid receptor (Giguère et al., 1987) also confirm the importance of region С for activation of gene transcription, since insertions which alter the spacing of the cysteines in this region either inactivate or drastically reduce the ability of the receptor to stimulate MMTV- CAT transcription. A point mutation in this region of a mouse nt~ glucocorticoid receptor, which changes an arginine to a histidine, reduces the ability of the receptor to bind tightly to the nucleus (Danielson et al., 1986). The integrity of this region is also essential for specific DNA binding in vitro. In the case of both the rat (Rusconi et al., 1986) and the human (Hollenberg et al., 1987) glucocorticoid receptors any deletions which remove part of this highly conserved region result in the loss of specific DNA binding using an in vitro DNA-binding assay. As discussed above, there is no apparent sequence homology within region A-B of the estrogen receptor between any of the members of the steroid and thyroid hormone receptor family. Insertion and deletion mutants within the human gluco¬ corticoid receptor have defined a region known as Tau 1 between amino acids 77 to 262 which is essential in order to activate MMTV-CAT transcription fully (Giguère et al., 1986; Hollenberg et al., 1987). Deletion mutants of the rat glucocorticoid receptor, within the region equivalent to the region A-B of the estrogen receptor, also appear to reduce the receptor's ability to activate MMTV-CAT transcription in transient assays and endogenous MMTV and a-l-acid glycoprotein (AGP) transcrip¬ tion in stably transformed cell lines (Miesfeld et al., 1987). In the case of both the rat and the human receptors the removal of N-terminal sequences extending close to the cysteine-rich region results in receptors which have approximately 10% of wild type activity. That some activity remains when this region is deleted suggests that whilst this region may not be absolutely essential for receptor function, it is clearly important for full activity. Therefore these results can be inteфreted as indicating either that this region plays a modulatory role (Hollenberg et al., 1987) or that the deletion of this region results in a loss of structural constraints provided by sequences located outside of the essential functional domains (Miesfeld et al., 1987). 2. Progesterone receptor An amino acid sequence comparison between the chick progesterone receptor and its human (Misrahi et al., 1987; A. Krust, U. Stropp, P. Kastner and P. Chambón, unpublished results) and rabbit (Loosfelt et al., 1986) counteфarts reveals two highly conserved regions. The cysteine-rich region С spanning from amino acid number 410 to 495 (see Fig. 4 and top of Fig. 16) exhibits a 100% conservation, whereas region E from amino acid 540 to the С terminus is 87% conserved, when compared with the progesterone receptor of mammals. These two regions are separated by a region D of lower homology (~60%). In contrast, the N-terminal region (termed A-B by analogy with the corresponding region of the estrogen