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Volume 272, Number 23, Issue of June 6, 1997 pp. 14592-14599
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Mutations in the 1,25-Dihydroxyvitamin D₃ Receptor Identifying C-terminal Amino Acids Required for Transcriptional Activation That Are Functionally Dissociated from Hormone Binding, Heterodimeric DNA Binding, and Interaction with Basal Transcription Factor IIB, in Vitro

(Received for publication, December 16, 1996, and in revised form, April 1, 1997)

Peter W. Jurutka , Jui-Cheng Hsieh , Lenore S. Remus , G. Kerr Whitfield , Paul D. Thompson , Carol A. Haussler , Jorge C. G. Blanco ^§ , Keiko Ozato ^§ and Mark R. Haussler

From the  Department of Biochemistry, College of Medicine, The University of Arizona, Tucson, Arizona 85724 and the ^§ Laboratory of Molecular Growth Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892

To investigate a potential ligand-dependent transcriptional activation domain (AF-2) in the C-terminal region of the human vitamin D receptor (hVDR), two conserved residues, Leu-417 and Glu-420, were replaced with alanines by site-directed mutagenesis (L417A and E420A). Transcriptional activation in response to 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) was virtually eliminated when either point mutant was transfected into several mammalian cell lines. Furthermore, both mutants exhibited a dominant negative phenotype when expressed in COS-7 cells. Scatchard analysis at 4 °C and a ligand-dependent DNA binding assay at 25 °C revealed essentially normal 1,25-(OH)₂D₃ binding for the mutant hVDRs, which were also equivalent to native receptor in associating with the rat osteocalcin vitamin D responsive element as a presumed heterodimer with retinoid X receptor. Glutathione S-transferase-human transcription factor IIB (TFIIB) fusion protein linked to Sepharose equally coprecipitated the wild-type hVDR and the AF-2 mutants. These data implicate amino acids Leu-417 and Glu-420, residing in a putative -helical region at the extreme C terminus of hVDR, as critical in the mechanism of 1,25-(OH)₂D₃-stimulated transcription, likely mediating an interaction with a coactivator(s) or a component of the basal transcriptional machinery distinct from TFIIB.

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