title: Reconstitution of the N-terminal transcription activation function of human mineralocorticoid receptor in a defective human glucocorticoid receptor.
abstract: 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 hMRDelta59-162, hMRDelta59-247, hMRDelta59-328, hMRDelta162-247, hMRDelta247-328, hMRDelta247-382, and hMRDelta328-382 with intact DNA-binding and hormone-binding domains. A second set of mutant receptors with unique BglII sites was generated to facilitate the isolations of fragments. Immunodetection with anti-hMR peptide antibodies and hormone-binding assays showed that the mutations did not affect the expression of the receptors or ability to bind aldosterone. Distribution of aldosterone binding activity of wild type and deletion mutants expressed in HeLa cells was predominantly nuclear. Furthermore, deletion of sequences between 59 and 390 did not affect DNA binding activity. Transfection studies with HeLa cells revealed a region around residue 247 that was crucial for normal receptor function. Deletion of amino acids 59-162 did not affect the transcriptional activity of the hMR. However, deletion of sequences 247-382 and 328-382 markedly decreased the transcription activation function. The induction of the reporter gene by the chimera hGRDelta71-262/hMR328-382 was 2-fold higher than with the wild type hGR, but 200-fold when compared with hGRDelta71-262, indicating that the AF-1 domain is located between positions 328 and 382 in the hMR.
authors.affiliation: Centre Recherche Hôtel-Dieu Québec and Laval University, Côte du Palais, Québec G1R 2J6, Canada. manjapra.govindan@crhdq.ulaval.ca
authors.forename: M V
authors.initials: MV
authors.lastname: Govindan
authors.forename: N
authors.initials: N
authors.lastname: Warriar
mesh_terms: D000450:Aldosterone / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D017354:Point Mutation; D018161:Receptors, Mineralocorticoid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D018161:Receptors, Mineralocorticoid; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D000450:Aldosterone