VITAMIN A AS PRECURSOR OF INTRACELLULAR SECOND MESSENGER

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Among the pleiotropic functions of retinol, the effects on the immune system are noteworthy, as reflected in the almost total dependence of B and T cells for growth and activation in culture on this retinoid. While searching for the mechanism of action, we have defined a metabolite of retinol, 14-hydroxy-retro-retinol (14HRR) that is the likely mediator of retinol effects in lymphocytes. The working hypothesis to be tested is that 14HRR acts as a second messenger molecule to transmit antigen- receptor generated activation signal(s) to cytoplasmic or nuclear receptor molecules. this hypothesis has been buoyed by the exciting finding that a close structural relative of 14HRR, anhydro-retro-retinol (ARR), competes for binding to similar target receptors and causes reversible inhibition of T cell activation and B cell growth. Transformed B cells cease to proliferate and die within 12 to 24 h with muM concentrations of ARR. Drawing on the previous body of work on 14HRR and on new observations with the antagonist ARR, we propose to continue the isolation and structural characterization of several new retinoids of mammalian and insect cells (Aim #1). 14HRR and 13,14 dihydroxy- retinol (DHR), a new retinoid and likely biosynthetic intermediate in 14HRR synthesis, will be synthesized to prove the structures and determine the stereochemical properties of the natural compounds and to produce 3H-labeled compounds for biological experimentation (Aim #2). The putative biosynthetic pathway from retinol via 13,14 epoxy-retinol and DHR to 14HRR will be investigated (Aim 3). 14HRR binding proteins in cytoplasm and/or nucleus will be identified by use of conventional protein fractionation in conjunction with assays for 14HRR binding and by use of an affinity matrix constructed with ARR. The identification of 14HRR binding proteins may open new vistas on the mechanism of action and suggest alternatives to the previously held (and still preferred) hypothesis that 14HRR acts as ligand for a nuclear transcription factor (Aim 4).