Metal binding 'finger' structures in the glucocorticoid receptor defined by site-directed mutagenesis.
The glucocorticoid receptor and the other members of the steroid receptor super-family share a highly conserved, cysteine-rich region which coincides with the DNA binding/transactivating domain. It has been postulated that this region is folded into two 'zinc finger' structures, similar to those originally reported for the transcription factor TFIIIA. The first potential finger domain contains four conserved cysteines and one conserved histidine, while the second contains five conserved cysteines. Using site-directed mutagenesis, we have analysed the consequences of altering the proposed finger-like structures. Our results show that most of the mutations affecting the conserved cysteines result in a total loss of glucocorticoid receptor function. In one important exception, however, a conserved cysteine (Cys500) is dispensable for glucocorticoid receptor activity and therefore cannot be involved in complexing a metal ion to form a finger structure. Moreover, the replacement of either Cys476 or Cys482 by His residues maintains partial in vivo activity of the glucocorticoid receptor, while their exchange for an alanine or serine residue, respectively, eliminates receptor function. These results support, at a genetic level, the involvement of cysteines of the glucocorticoid receptor DNA binding domain in metal ion complexation and define the candidate residues involved in such coordination.
Severne
Y
Y
Institut für Molekularbiologie II, Universität Zürich, Switzerland.
Wieland
S
S
Schaffner
W
W
Rusconi
S
S
D000595:Amino Acid Sequence; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D015032:Zinc / Q000378:metabolism*
D011965:Receptors, Glucocorticoid; D006639:Histidine; D015032:Zinc; D003545:Cysteine