title: Molecular cloning, functional characterization, and evolutionary analysis of vitamin D receptors isolated from basal vertebrates.
abstract: The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication (3R) specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC50 values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D3 acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D3 throughout vertebrate evolution that may have been driven by changes in protein-protein interactions between VDR and essential coregulators.
authors: Program in Environmental and Molecular Toxicology, Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina, United States of America.
authors: Erin M
authors: EM
authors: Kollitz
authors: Bioinformatics Research Center, North Carolina State University, Raleigh, North Carolina, United States of America.
authors: Guozhu
authors: G
authors: Zhang
authors: Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina, United States of America.
authors: Mary Beth
authors: MB
authors: Hawkins
authors: Department of Basic Medical Sciences, University of Arizona College of Medicine, Phoenix, Arizona, United States of America.
authors: G Kerr
authors: GK
authors: Whitfield
authors: Bioinformatics Research Center, North Carolina State University, Raleigh, North Carolina, United States of America; Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina, United States of America.
authors: David M
authors: DM
authors: Reif
authors: Program in Environmental and Molecular Toxicology, Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina, United States of America; Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina, United States of America.
authors: Seth W
authors: SW
authors: Kullman
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D019295:Computational Biology; D024202:Electrophoretic Mobility Shift Assay; D019143:Evolution, Molecular; D005399:Fishes / Q000235:genetics*; D016013:Likelihood Functions; D008957:Models, Genetic; D008969:Molecular Sequence Data; D048428:Petromyzon / Q000235:genetics*; D010802:Phylogeny; D011485:Protein Binding; D025941:Protein Interaction Mapping; D018167:Receptors, Calcitriol / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012860:Skates, Fish / Q000235:genetics*; D013045:Species Specificity; D020798:Two-Hybrid System Techniques
chemical_list: D018167:Receptors, Calcitriol