Vitamin A is an essential nutrient required for growth, reproduction, differentiation of epithelial tissues and vision. The localization of cellular retinol binding protein II (CRBP II) exclusively to the small intestine in the adult animal suggests that this protein is uniquely adapted to the intestinal absorption and metabolism of retinol. The Principal Investigator proposes to study the structure, function, and regulation of CRBP II. Triple resonance multidimensional nmr techniques will be employed to study the structure and dynamics of CRBP II in solution. Regions of the protein which undergo ligand induced changes in conformation and flexibility will be identified, in order to gain further insights as to how retinol enters and is released from the binding cavity. CRBP II has been postulated to play an important role in the uptake and intracellular trafficking of retinol between different metabolic pathways within the enterocyte, however this remains to be established in vivo. The Principal Investigator proposes to construct a targeting vector for the disruption of the CRBP II gene in embryonal stem cells by homologous recombination, and to examine vitamin A homeostasis in the CRBP II-/- mice compared to wild-type littermates. The proximal region of the mouse and rat CRBP II genes contain cis-acting elements which are activatable by (retinoid X receptor) RXR homodimeric and heterodimeric complexes. The investigator will analyze the role of RXR dependent signaling pathways in the regulation of the endogenous human CRBP II gene in differentiated Caco-2 cells using retinoid receptor selective ligands and a well characterized potent dominant negative mutant RAR alpha 403. The Principal Investigator will also target transgenic expression of the dominant negative to villus-associated epithelial cells to the intestine under the control of the intestinal fatty acid binding protein FABPi promoter, to determine the role of RXR-dependent signaling pathways in regulating expression of the mouse CRBP II gene as well as other intestinal genes in vivo.
