RECONSTITUTION OF THE N-TERMINAL TRANSCRIPTION ACTIVATION FUNCTION OFHUMAN MINERALOCORTICOID RECEPTOR IN A DEFECTIVE HUMAN GLUCOCORTICOID RECEPTOR

N-terminal sequences involved in transcription activation by the human mineralocorticoid receptor (hMR) have yet to be defined, We have addr essed this issue and generated overlapping internal deletion mutants h MR Delta(59-162), hMR Delta(59-247), hMR Delta(59-328), hMR Delta(162- 247), hMR Delta(247-328), hMR Delta(247-382), and hMR Delta(328-382), with intact DNA-binding and hormone-binding domains. A second set of m utant receptors with unique BglII sites was generated to facilitate th e isolations of fragments. Immunodetection with anti-hMR peptide antib odies and hormone-binding assays showed that the mutations did not aff ect the expression of the receptors or ability to bind aldosterone, Di stribution of aldosterone binding activity of wild type and deletion m utants expressed in HeLa cells was predominantly nuclear, Furthermore, deletion of sequences between 59 and 390 did not affect DNA binding a ctivity. Transfection studies with HeLa cells revealed a region around residue 247 that was crucial for normal receptor function. Deletion o f amino acids 59-162 did not affect the transcriptional activity of th e hMR. However, deletion of sequences 247-382 and 328-382 markedly dec reased the transcription activation function. The induction of the rep orter gene by the chimera hGR Delta(71-262)/hMR(328-382) was 2-fold hi gher than with the wild type hGrR, but 200-fold when compared with hGR Delta(71-262), indicating that the AF-1 domain is located between pos itions 328 and 382 in the hMR.