Functional Diversification of the Vitamin D Receptor (VDR) with Increasing Genome Complexity in Vertebrates

The vertebrate genome is a result of two rapid and successive rounds of genome duplication (1R and 2R). Teleost fish maintain a greater number of nuclear receptors (NRs) compared to other vertebrates due to a third genome duplication specific to their lineage (3R). The retention of multiple nuclear receptor pairs in teleosts provides a unique opportunity to gain insight into how receptor paralogs evolve through specific evolutionary processes. This dissertation examines the functional diversification of the vitamin D receptor (VDR) with increasing genome complexity within teleost and vertebrate evolution. VDR function is assessed in response to 1alpha, 25-dihydroxyvitamin D 3 (1, 25D3) and lithocholic acid (LCA), a toxic secondary bile acid.;Chapter 1 examines the hypothesis that teleost VDRalpha and VDRbeta paralogs are derived from the 3R event, and that retention of duplicate VDRs is likely due to sub- or neofunctionalization. VDR paralogs were cloned from the Japanese medaka (Oryzias latipes) and the zebrafish ( Danio rerio). We demonstrate that high affinity binding to 1, 25D 3 has been conserved between paralogs, however transactivational efficacy varies significantly. Subsequent studies demonstrate that VDRalpha paralogs exhibit preferential DNA binding compared to the VDRbeta paralogs, and demonstrate differential protein-protein interactions between paralogs and essential co-regulators including RXR and the SRC family of nuclear receptor co-activators. We speculate that the observed functional differences are due to subtle conformational differences between paralogs.;Chapter 2 examines the hypothesis that the receptor-ligand partnership between VDR and 1, 25D3 is ancient and traceable to basal extant vertebrate species. Basal VDRs were cloned from the sea lamprey ( Petromyzon marinus) a jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish; and the Senegal bichir ( Polypterus senegalus), a primitive 2R ray-finned fish. Similar to the teleost VDR paralogs, we demonstrate that basal VDR orthologs maintain high affinity for 1, 25D3, maintain similar DNA binding characteristics, and that this ligand serves as a potent transactivational agonist with these receptors. However, transactivational efficacy varies significantly between VDR orthologs. Subsequent studies indicate protein-protein interactions between basal VDRs and essential co-regulators exhibit significant differences between VDR orthologs. Results from a cluster analysis support the notion that the observed functional differences are predominantly driven through differential interactions between receptors and their coregulators. Results from the cluster analysis suggest that the basal VDRs function similar to the VDRbeta paralogs, while the VDRalpha paralogs function more similar to human VDR. Our results provide further evidence a functional divergence between the teleost VDR paralogs, and support the hypothesis that the 1, 25D3 -- VDR partnership is ancient.;Chapter 3 examines the hypothesis that that the ligand-receptor partnership between VDR and LCA is a result of exaptation. It has been speculated that the VDR-lithocholic acid partnership in mammals resulted from an adaptive process associated with the synthesis and detoxification of the bile acid in higher vertebrates. However, the evolutionary history of this partnership is not well understood due to the lack of data from non-mammalian vertebrates. We demonstrate that LCA and 3-Keto both function as full agonists only with human VDR and the teleost VDRalpha paralogs. Interaction between the bile acids and VDRbeta paralogs did not facilitate transactivation or RXR heterodimerization, and display attenuated coactivator recruitment. Similarly, VDR from basal vertebrates were not able to mediate any response to LCA or 3-Keto beyond ligand binding. Bioinformatics analysis suggests that functional differences between VDRs are driven though differential interactions between VDRs and essential coactivators. Our results suggest that the ability of LCA to function as a VDR ligand evolved before the bile acid itself, most likely though a process of exaptation followed by co-option once the need to detoxify LCA arose in higher vertebrates.