The long term objective of this applications are to understand the movement and metabolism of compounds of the vitamin A family, the retinoids. The retinoids play essential roles in embryogenesis, morphogenesis, and maintenance of many tissues of the mature animal, by regulating the expression of specific genes. Retinoic acid is perhaps the major hormonal form of vitamin A but much is still to be learned about the mechanism, site, and control of its production. Specific binding our career proteins are involved in the movement and metabolism of retinoids. For retinoic acid production, deliver of its precursor, retinol, by the extracellular protein, retinol-binding protein (RBP), is important. For production and export of retinoic acid from the cells that snythesize it, the intracellular protein, cellular retinoic acid-binding protein, type two [CRABP(II)] appears to be important. In this proposal, several model systems from the pseudopregnant rat will be used to study the production of retinoic acid for function as a hormone. Results will be contrasted to retinoic acid production by the liver and kidney, hypothesized here to be for the purpose of retinol catabolism. The work proposed will: 1. Characterize the metabolic pathway and enzymology that leads to the production of retinoic acid from retinol provided to cultured cells as a complex with RBP. Uterine and ovarian cells that synthesize retinoic acid will be the source for determining the pathway and enzymology by standard biochemical techniques. 2. Characterize the role that CRABP(II) plays in the cell's ability to synthesize and secrete retinoic acid. Cultured cells will be deprived of CRABP(II) by antisense technology to study the effect on retinoic acid production/export. 3. Identify the mechanism of delivery of retinol, from the retinol-RBP complex, to the epithelial cells that make retinoic acid. Possible internalization of RBP will be characterized by following labelled protein. 4. Seek to establish the existence of a specific membrane transporter that controls the secretion of retinoic acid from the cells that synthesize it. If identified by uptake studies, expression cloning will be utilized to isolate its cDNA.
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