Mapping the domains of the interaction of the vitamin D receptor and steroid receptor coactivator-1

The vitamin D receptor (VDR) binds to the vitamin D response element (VDRE) and mediates the effects of the biologically active form of vitamin D, 1,25-dihydroxyvitamin D-3 [1,25-(OH)(2)D-3], on gene expression. The VDR binds to the VDRE as a heterodimeric complex with retinoid X receptor, In the present study, we have used a yeast two-hybrid system to clone complementary DNA that codes for VDR-interacting protein(s). We found that the human steroid receptor coactivator-1 (SRC-1) interacts with the VDR in a ligand-dependent manner, as demonstrated by beta-galactosidase production. The interaction of the VDR and the SRC-1 takes place at physiological concentrations of 1,25(OH)(2)D-3. A 48.2-fold stimulation of beta-galactosidase activity was observed in the presence of 10(-10) M 1,25-(OH)(2)D-3. In addition, a direct interaction between the ligand-activated glutathione-S-transferase-VDR and S-35-labeled SRC-1 was observed in vitro. Deletion-mutation analysis of the VDR established that the ligand-dependent activation domain (AF-2) of the VDR is required for the interaction with SRC-1, One deletion mutant, pGVDR-(1-418), bound the ligand but failed to interact with the SRC-1, whereas another deletion mutant, pGVDR-(1-423), bound the ligand and interacted with the SRC-1, We demonstrated that all the deletion mutants were expressed as analyzed by a Gal(4) DNA-binding domain antibody. Deletion mutation analysis of the SRC-1 demonstrated that 27 amino acids (DPCNTNPTPMTKATPEEIKLEAQSQFT) of the SRC-1 are essential for interaction with the AF-2 motif of the VDR.