Interdomain interactions in the mineralocorticoid receptor.
The potential for interaction between the N-terminal domain and the C-terminal region (hinge and ligand-binding domain) of the mineralocorticoid receptor (MR) was examined using the mammalian-2-hybrid assay. The MR C-terminal region was fused to the GAL4 DNA-binding domain (GAL4-MRC). To examine if the AF-2 is involved in the interaction, as has been reported for other steroid hormone receptors, it was inactivated by point mutation (E962A). The N-terminal domain was fused to the VP16 transactivation domain (VP16-MRNT). In the mammalian-2-hybrid assay both GAL4-MRC and GAL4-MRC(E962A) interact with VP16-MRNT in an aldosterone-dependent manner. The GAL4-MRC(E962A) construct was used in subsequent experiments to examine the AF-2-independent N/C-interaction. The MR antagonist spironolactone inhibits the aldosterone-mediated association of the two domains. GAL4-MRC(E962A) interacts weakly with the GR or AR N-terminal domains in the presence of aldosterone. No dimerization between GAL4-MRC(E962A) and VP16-MRC is observed. Interestingly, cortisol produces a much weaker N/C-interaction than aldosterone, and it is possible that the N/C-interaction may contribute to observed functional differences in the MR bound to the two ligands.
Rogerson
Fraser M
FM
Prince Henry's Institute of Medical Research, P.O. Box 5152, Clayton, Vic. 3168, Australia. fraser.rogerson@med.monash.edu.au
Fuller
Peter J
PJ
D000450:Aldosterone / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D019281:Dimerization; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D018161:Receptors, Mineralocorticoid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013148:Spironolactone / Q000494:pharmacology; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
D018161:Receptors, Mineralocorticoid; D011993:Recombinant Fusion Proteins; D013148:Spironolactone; D000450:Aldosterone; D006854:Hydrocortisone