Vitamin D-dependent suppression of human atrial natriuretic peptide gene promoter activity requires heterodimer assembly

Crystallographic structures of the ligand-binding domains for the retinoid X (RXR) and estrogen receptors have identified conserved surface residues t hat participate in dimer formation. Homologous regions have been identified in the human vitamin D receptor (hVDR). Mutating Lys-386 to Ala (K386A) in hVDR significantly reduced binding to glutathione S-transferase-RXR alpha in solution3 whereas binding of an I384R/Q385R VDR mutant was almost undete ctable. The K386A mutant formed heterodimers with RXR alpha on DR-3 (a dire ct repeat of AG-GTCA spaced by three nucleotides), whereas the I384R/Q385R mutant completely eliminated heterodimer formation. Wild type hVDR effected a 3-fold induction of DR-3-dependent thymidine kinase-luciferase activity in cultured neonatal rat atrial myocytes, an effect that was increased to 8 -9-fold by cotransfected hRXR alpha. Induction by K386A, in the presence or absence of RXRa, was only slightly lower than that seen with wild type VDR . On the other hand, I384R/Q385R alone displayed no stimulatory activity an d less than 2-fold induction in the presence of hRXR alpha. Qualitatively s imilar findings were observed with the negative regulation of the human atr ial natriuretic peptide gene promoter by these mutants. Collectively, these studies identify specific amino acids in hVDR that play a critical role in heterodimer formation and subsequent modulation of gene transcription.