Distinct retinoid X receptor activation function-2 residues mediate transactivation in homodimeric and vitamin D receptor heterodimeric contexts

The vitamin D receptor (VDR) stimulates transcription as a 1,25-dihydroxyvi tamin D-3 (1,25(OH)(2)D-3)-activated heterodimer with retinoid X receptor ( RXR). RXR also forms homodimers to mediate 9-cis retinoic acid (9-cis RA)-i nduced gene expression. Both receptors possess a C-terminal hormone-depende nt activation function-2 (AF-2), a highly conserved region that binds coact ivators to transduce the transcriptional signal. By replacing single amino acids within the AF-2 of human RXR alpha (hRXR alpha) or mouse RXR beta (mR XR beta), the contribution of these residues to transactivation by the RXR- VDR heterodimer and the RXR-RXR homodimer was evaluated. In 9-cis RA-respon sive homodimers, the second and fourth positions of the AF-2 (leucine and g lutamate respectively) are essential. However, in the context of an RXR-VDR heterodimer activated by 1,25(OH)(2)D-3, alteration of these two RXR resid ues has little effect. Instead, AF-2 residues located towards the C-terminu s, such as the penultimate position (L455 in hRXR alpha or L441 in mRXR bet a), are crucial for RXR-VDR heterodimers. Indeed, L455A mutant RXR exerts a dominant negative effect on RXR-VDR transcriptional responsiveness to 1,25 (OH)(2)D-3. Further experiments with a mutant hRXR alpha (F313A) which elic its 9-cis RA-independent transactivation as a homodimer demonstrate that wh en heterodimerized with VDR, this RXR mutant is incapable of activating the RXR-VDR heterocomplex in the absence of the VDR ligand. Taken together, th ese results indicate that RXR is a subordinate, yet essential transcription al partner in RXR-VDR-mediated activation of gene expression. Furthermore, a functional switch in RXR AF-2 signaling occurs between RXR residues in th e homodimeric versus the heterodimeric states, likely reflecting different interactions between subregions of the AF-2 and coactivator(s).