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 vita min 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 syste m to clone complementary DNA that codes for VDR-interacting protein(s) . We found that the human steroid receptor coactivator-1 (SRC-1) inter acts with the VDR in a ligand-dependent manner, as demonstrated by bet a-galactosidase production. The interaction of the VDR and the SRC-1 t akes place at physiological concentrations of 1,25(OH)(2)D-3. A 48.2-f old stimulation of beta-galactosidase activity was observed in the pre sence 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-la beled SRC-1 was observed in vitro. Deletion-mutation analysis of the V DR established that the ligand-dependent activation domain (AF-2) of t he 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 an d interacted with the SRC-1, We demonstrated that all the deletion mut ants were expressed as analyzed by a Gal(4) DNA-binding domain antibod y. Deletion mutation analysis of the SRC-1 demonstrated that 27 amino acids (DPCNTNPTPMTKATPEEIKLEAQSQFT) of the SRC-1 are essential for int eraction with the AF-2 motif of the VDR.