A HIGHLY CONSERVED REGION IN THE HORMONE-BINDING DOMAIN OF THE HUMAN VITAMIN-D-RECEPTOR CONTAINS RESIDUES VITAL FOR HETERODIMERIZATION WITHRETINOID-X-RECEPTOR AND FOR TRANSCRIPTIONAL ACTIVATION

Residues located between amino acids 244 and 263 in the human vitamin D receptor (hVDR) show extensive homology with other members of the st eroid/thyroid/retinoid hormone receptor superfamily. The corresponding region of the glucocorticoid receptor has been shown to interact with the 90-kilodalton heat shock protein (hsp90), yet hVDR does not appea r to bind to hsp90. Herein we report a study of hVDR in which the func tional role of five conserved residues was tested by replacing Phe-244 , Lys-246, Leu-254, Gln-259, and Leu-262 with glycines by site-directe d mutagenesis. Initial screening of these mutants indicated that all w ere significantly impaired in their ability to activate transcription from a vitamin D-responsive reporter construct when expressed in trans fected VDR-deficient COS-7 cells. Further characterization revealed tw o classes of mutants: the predominant class binds the 1,25-dihydroxyvi tamin D-3 ligand normally but is defective in its ability to form a he terodimeric complex with the retinoid X receptor (RXR) on a vitamin D responsive element (VDRE). A second unique class, represented by a sin gle mutant at Lys-246, is normal both with respect to ligand binding a nd complex formation but still very impaired in transactivation abilit y. The distinction between these two classes was confirmed by the demo nstration that a member of the first class, with a mutation at Gln-259 , could be restored to near wild type transactivation ability by suppl ying excess RXR, while the Lys-246 mutant could not be so rescued. We therefore conclude that the primary function of this conserved domain in hVDR is the mediation of heterodimerization with RXR, leading to VD RE binding and transactivation. The possibility also exists that the L ys-246 mutant may be impaired in a step of transactivation that is dis tal to complex formation with RXR on the VDRE, perhaps in interactions with the transcriptional machinery itself.