Mapping the Domains of the Interaction of the Vitamin D Receptor and Steroid Receptor Coactivator-1

• Published in: Molecular endocrinology (Baltimore, Md.) Md.), 1998-01, Vol.12 (1), p.57-65
• Main Authors: Gill, Rajbir K, Atkins, Loretta M, Hollis, Bruce W, Bell, Norman H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; beta-Galactosidase - genetics; Calcitriol - metabolism; DNA, Complementary - isolation & purification; Gene Expression Regulation; Gene Library; Histone Acetyltransferases; Humans; Kidney; Ligands; Molecular Sequence Data; Nuclear Receptor Coactivator 1; Peptide Mapping; Protein Structure, Tertiary; Receptors, Calcitriol - genetics; Receptors, Calcitriol - isolation & purification; Receptors, Calcitriol - metabolism; Receptors, Steroid - metabolism; Recombinant Fusion Proteins - genetics; Saccharomyces cerevisiae; Transcription Factors - genetics; Transcription Factors - isolation & purification; Transcription Factors - metabolism
• ISSN: 0888-8809; 1944-9917
• DOI: 10.1210/mend.12.1.0048

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 β-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 β-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 (DPCNTNPTPMTKATPEEIKLEAQS-QFT) of the SRC-1 are essential for interaction with the AF-2 motif of the VDR.