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-dihydroxyvitamin D₃ (1,25(OH)₂D₃)-activated heterodimer with retinoid X receptor (RXR). RXR also forms homodimers to mediate 9-cis retinoic acid (9-cis RA)-induced gene expression. Both receptors possess a C-terminal hormone-dependent activation function-2 (AF-2), a highly conserved region that binds coactivators to transduce the transcriptional signal. By replacing single amino acids within the AF-2 of human RXRα (hRXRa) or mouse RXRβ (mRXRβ), the contribution of these residues to transactivation by the RXR-VDR heterodimer and the RXR-RXR homodimer was evaluated. In 9-cis RA-responsive homodimers, the second and fourth positions of the AF-2 (leucine and glutamate respectively) are essential. However, in the context of an RXR-VDR heterodimer activated by 1,25(OH)₂D₃, alteration of these two RXR residues has little effect. Instead, AF-2 residues located towards the C-terminus, such as the penultimate position (L455 in hRXRα or L441 in mRXRβ), are crucial for RXR-VDR heterodimers. Indeed, L455A mutant RXR exerts a dominant negative effect on RXR-VDR transcriptional responsiveness to 1,25(OH)₂D₃. Further experiments with a mutant hRXRα (F313A) which elicits 9-cis RA-independent transactivation as a homodimer demonstrate that, when heterodimerized with VDR, this RXR mutant is incapable of activating the RXR-VDR heterocomplex in the absence of the VDR ligand. Taken together, these results indicate that RXR is a subordinate, yet essential transcriptional partner in RXR-VDR-mediated activation of gene expression. Furthermore, a functional switch in RXR AF-2 signaling occurs between RXR residues in the homodimeric versus the heterodimeric states, likely reflecting different interactions between subregions of the AF-2 and coactivator(s).