SUPPRESSION OF ANP GENE-TRANSCRIPTION BY LIGANDED VITAMIN-D-RECEPTOR - INVOLVEMENT OF SPECIFIC RECEPTOR DOMAINS

We showed previously that liganded vitamin D receptor (VDR) effects a suppression of human atrial natriuretic peptide (hANP) gene-promoter a ctivity in cultured neonatal rat atrial myocytes. In the present study , we have attempted to identify the structural domains of the VDR that are involved in mediating this suppression. We examined the effects o f a series of VDR mutants on a cotransfected hANP promoter-driven chlo ramphenicol acetyltransferase (CAT) reporter. Neither the native VDR n or any of the mutants tested displayed inhibitory activity in the abse nce of the 1,25-dihydroxyvitamin D-3 (VD3) ligand. Delta 134, a deleta nt harboring solely the DNA binding region of the VDR, and L254G, a mu tant shown to be defective in retinoid X receptor (RXR) heterodimer fo rmation in other systems, were as effective as the native VDR in reduc ing promoter activity. HBD, a deletant containing only the hormone-bin ding domain of the VDR, and K246G, a point mutant that is defective in the activation function of the receptor, did not attenuate reporter a ctivity. A similar activity profile was displayed when a positively re gulated promoter containing a direct-repeat vitamin D responsive eleme nt (DR3-CAT) was examined in these cells. Liganded VDR, the Delta 134 mutant, and liganded L254G effected increases in DR3-CAT activity of 2 .5-, 2-, and 4-fold, respectively. Two nonhypercalcemic analogues of V D3 (RO 23-7553 and RO 25-6760) displayed the same inhibitory activity as VD3. These studies suggest that the inhibition of hANP promoter act ivity requires both the DNA binding and activation functions of the re ceptor but does not appear to require formation of a classic RXR alpha -VDR heterodimer.