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 D3 [1,25-(OH)2D3], 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)2D3. A 48.2-fold stimulation of beta-galactosidase activity was observed in the presence of 10(-10) M 1,25-(OH)2D3. In addition, a direct interaction between the ligand-activated glutathione-S-transferase-VDR and 35S-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 Gal4 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.
Gill
R K
RK
Department of Medicine, Medical University of South Carolina, Department of Veterans Affairs Medical Center, Charleston 29401-5799, USA.
Atkins
L M
LM
Hollis
B W
BW
Bell
N H
NH
D000595:Amino Acid Sequence; D002117:Calcitriol / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D005786:Gene Expression Regulation; D015723:Gene Library; D051548:Histone Acetyltransferases; D006801:Humans; D007668:Kidney; D008024:Ligands; D008969:Molecular Sequence Data; D056920:Nuclear Receptor Coactivator 1; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D018167:Receptors, Calcitriol / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics
D018076:DNA, Complementary; D008024:Ligands; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1; D001616:beta-Galactosidase; D002117:Calcitriol