Identification of an autonomous transactivation domain in helix H3 of the vitamin D receptor.
The vitamin D receptor (VDR) contains an alpha-helical, ligand-inducible activation function (AF-2) at the COOH-terminus of the ligand-binding domain (LBD). In this study, a second distinct activation domain was identified in the VDR LBD. Using a yeast-based system to screen a random mutant library of GAL4-VDR (93-427), a mutant GAL4-VDR fusion protein with constitutive transcriptional activity was isolated. Sequence analysis identified a C to T transition that introduced a stop codon at glutamine 239 eliminating a large portion of the LBD, including the AF-2 domain. The GAL4-VDR (93-238) mutant exhibited ligand-independent transactivation activity both in yeast and in mammalian cells. Deletion analysis defined a minimal activation domain within helix H3 between D195 and I 238 in the VDR. An aspartic acid residue (D232) within helix H3 was essential for the autonomous transactivation activity since altering this residue to an alanine or an asparagine dramatically reduced its transactivation potential. Expression of the minimal helix H3 activation domain interfered with ligand-activated transcription by full-length VDR suggesting that helix H3 interacts with limiting cellular factors important for VDR-activated transcription. Consequently, we have identified a novel activation domain in helix H3 of the VDR that apparently plays an important role in 1,25-(OH)(2)D(3)-activated transcription.
Kraichely
D M
DM
St. Louis University Health Sciences Center, Department of Pharmacological and Physiological Science, St. Louis, Missouri 63104, USA.
Nakai
Y D
YD
MacDonald
P N
PN
D002117:Calcitriol / Q000494:pharmacology; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection; D015003:Yeasts
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D002117:Calcitriol