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). (VDR). This nuclear protein belongs to a large superfamily, which includes receptors for steroids, retinoids, and thyroid hormone (1). As with other members of this superfamily, the VDR possesses an N-terminal DNA binding domain (DBD) that contains two zinc finger DNA binding motifs (2). The C-terminal portion of the VDR contains a ligand binding domain (LBD) that associates with the 1,25-(OH) 2 D 3 hormone (3). This binding initiates a presumed conformational change in VDR that enhances its interaction with any one of several isoforms of retinoid X receptor (RXR) to form a heterodimer, the apparent active species in recognizing and binding with high affinity to vitamin D-responsive elements (VDREs) located in the upstream promoter region of genes regulated by 1,25-(OH) 2 D 3 (4, 5). In addition to binding 1,25-(OH) 2 D 3 , the C-terminal LBD of VDR contains residues necessary for heterodimerization with RXRs (6, 7). When the activated, ligand-bound VDR:RXR heterodimer is associated with the VDRE, it stimulates the transcription of downstream target genes (8). Several VDREs have been characterized to date, with the prototypical sequence consisting of an imperfect direct repeat of six nucleotide bases, GGGTGA, separated by a 3-base pair spacer (9 -13). VDR has been shown to contact the base pairs in the 3Ј half-site of the VDRE, while RXR interacts with the 5Ј half-site (14).The precise mechanism of transcriptional regulation by the activated VDR:RXR heterodimer is not well understood. Functional analyses of members of the nuclear receptor superfamily, including truncation and point mutagenesis studies, have demonstrated the presence of at least two major domains involved in receptor-mediated transcriptional stimulation. The N-terminal regions of several nuclear receptors contain a constitutive activation domain referred to as AF-1 (also designated 1) (15-17) that can be linked to heterologous DNA binding domains to create functional transcription factors (18 -20). Moreover, fusion of the large C-terminal domain of various receptors to the GAL4 or glucocorticoid receptor (GR) DBD produces a chimeric protein capable of activating transcription in response to the cognate ligand (18,21,22). A subdomain of this hormonedependent, C-terminal activation function is known as AF-2 (also termed 4) (21,(23)(24)(25). Unlike the N-terminal AF-1 transcription domain, the C-terminal AF-2 activation unit exhibits a higher degree of homology among the nuclear receptor superfamily, suggesting a common or related mechanism may be involved in ligand-mediated gene regulation. In one study (26), the AF-2 region of the thyroid hormone receptor (TR) was reported to interact with the basal transcription factor IIB (TFIIB), implying that association of nuclear receptors wi...