N-terminal sequences involved in transcription activation by the human mineralocorticoid receptor (hMR) have yet to be defined. We have addressed this issue and generated overlapping internal deletion mutants hMR⌬ 59 In humans, the biologically active mineralocorticoid aldosterone maintains the homeostasis of ion balance principally in the kidney, but also in the gut, salivary, and sweat glands. The evolution of the modular structure of nuclear receptors into distinct domains is largely the consequence of complex requirements that arose during cellular growth and development. The structures of the human mineralocorticoid receptor (hMR) 1 and the human glucocorticoid receptor (hGR) are very similar. The N-terminal region in this family of nuclear receptors is of variable length and contains a transactivation function, the AF-1 (1). In the absence of a hormone-binding domain (HBD), this region is constitutively active. The N-terminal regions of hGR and hMR have only 15% homology, and it is this domain that is responsible for differences in target gene specificity.In the hGR, the N-terminal activation domain is 185 amino acids long and a 58-amino acid peptide that is almost as active as the intact region has been identified (2). The experiments with hGR were, however, performed with constructs containing segments of the hGR expressed in yeast and not in mammalian cells, and the cooperative function of the hGR HBD was not taken into consideration in these analyses. Such regions in the hMR remain to be defined. Our experiments were performed in animal cells in culture, and the intact DNA-binding domain (DBD) and HBD of the hMR or hGR were used in transfection studies.Although 94% of the 68 amino acids in the centrally located DBD of hMR and hGR are identical, the HBD has only 57% homology. The hMR contains an additional 24 amino acids including a sequence of 4 glutamines and 8 prolines encoded by repetitive nucleotide elements (1). The C terminus of the HBD also contains a hormone-dependent transcription activation function (AF-2).In this paper, we have identified hMR sequences crucial for the transactivation function by first generating receptor mutants with unique BglII sites. In order to identify the hMR sequences capable of conferring activity upon a hGR that lacks transcriptional activity, we have constructed a series of hGR⅐hMR chimeras. To map the exact domain responsible for transactivation function of hMR, the chimeras were introduced into receptor-deficient CV-1 cells and sequences in the hMR (amino acids 328 -382) were identified. MATERIALS AND METHODSDNA Constructs-Cloning was performed by standard procedure (3). MMTV-CAT and the eukaryotic expression plasmids for various steroid receptors were constructed as described previously (4, 5). The hMR⌬ 59 -328 was created after digesting the cDNA with ScaI-ScaI. Subsequent religation generated an uninterrupted 715-amino acid sequence in an open reading frame. Similarly, digestion of hMR cDNA with BalI (amino acids 148/149 and 390/391) and religation generated...

To understand the function of cysteines, we have substituted cysteines 638, 643, and 665 by serine in the hormone-binding domain (HBD) of the human glucocorticoid receptor (hGR). In hormone-binding assays using [3H]dexamethasone, hGR C643S and hGR C665S exhibited wild type receptor Kd of 2.5 nM and hGR C665SM666L displayed a Kd of 3.7 nM, while hGR C638S exhibited a Kd of 162 pM, a 15-fold higher affinity. The affinity of hGR C638S for RU486 was 10-fold higher, and the mutants C643S and C665S bound RU486 with a 10-fold lower affinity when compared to wild type GR. While C665S bound aldosterone with very high relative affinity, the double mutant C665SM666L failed to bind aldosterone. The expression of wild type, mutant, and truncated hGRs in vitro showed an identical level of expression of the cloned receptors. Similar levels of expression of the receptors were observed in transfected cells, both by immunoprecipitation and by Western blotting. Transcription activation of the chimeric reporter gene mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) with hGR C638S was 4-fold higher than the level observed with wild type hGR in the presence of dexamethasone. In the presence of RU486, hGR C638S induced MMTV-CAT 25-fold compared to the highest levels observed with wild type hGR and RU486. Even though the hGR C665S stimulated transcription with aldosterone, hGR C665SM666L did not. DNA-receptor interaction analyses by gel mobility shift assay demonstrated that the increased transactivation potential of hGR C638S was due to its intense interaction with DNA. These findings suggest that C638 and C665 are involved in maintaining specificity to glucocorticoids.

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