The long-term goals are to understand the roles of specific binding proteins in vitamin A absorption, transport, and metabolism. A novel retinoic acid (RA) synthesizing system, co-expressed with cellular retinoic acid binding protein (II) (CRABP(II), has been identified in the female reproductive system and in human mammary epithelium. CRABP (II) and cellular retinoic acid binding protein (CRABP) have been demonstrated to be associated with the mitochondria. Specific studies to build on these observations are: (1) to determine the distribution/regulation of this and other RA synthesizing systems in rat tissues, and to characterize the RA synthesizing enzymes in human mammary epithelium to examine defects in tumororigenic cell lines; (2) to delineate the novel mechanisms of mitochondria association of the two retinoic acid binding proteins; (3)to access the role of mitochondria in retinoid metabolism; and (4) to reveal the participation of CRABP (II) in these processes. Experimental designs and methods are: (1) sites of expression of CRABP (II) and RA-synthesizing enzymes I epithelial linings of rat tissues will be revealed by immunohistochemistry and in situ hybridization. The effect of vitamin A deficiency on expression will be examined. (2) Sequences involved in mitochondrial association will be identified for CRABP and CRABP(II). Alanine-scanning mutagenesis will define the residues essential for association. Mitochondrial association will be followed by confocal microscopy of the pattern of immunofluorescence of proteins or of peptide fusions with green fluorescent protein for FLAG tag. Sequences established as important will be chemically synthesized with a photoactivatable group and attached biotin, incubated with mitroochondria and cell extracts and cross-linked to allow identification of proteins involved in recognition. (3) Appropriate mitochondria will be tested for ability to metabolize retinoids. Metabolities will be identified, including possible novel metabolities that might served as ligands for the retinoic acid nuclear receptors. (4) CRABP(II) will be mutated to ablate targeting to the mitrochondria to see if this """"""""loss of function"""""""" affects retinoid metabolism and movement in cells normally expressing this protein. These studies will increase our understanding of the essential role of retinoids in maintaining many epithelia, particularly epithelial prone to pathological conditions such as cancer.
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