Vitamin A is an indispensable factor in the diet. Retinoic acid, a naturally occuring biologically active form of vitamin A, plays an important role in modulating the growth and differentiation of both normal and transformed cells. The biological effects of retinoic acid appear to be modulated via retinoic acid binding proteins. These proteins include the small cellular retinoic acid binding proteins (CRABP-I and CRABP-II) and the nuclear retinoic acid receptors ( -RAR, beta-RAR, -RAR and RXR). Each of these retinoic acid binding proteins display an unique pattern of expression and appear to display differences in their affinity and specificity for retinoic acid and related retinoids. Little information is available concerning the exact mechanism of how retinoic acid binds to each of these retinoic acid binding proteins. Therefore in this grant application we wish (1) to predict the retinoic acid binding site of CRABP-I and CRABP-II using molecular modeling techniques; (2) to experimentally test the validity of the predicted retinoic acid binding site of CRABP-I and CRABP-II using site-directed mutagenesis and (30) to overexpress and purify wild type CRABP-I and CRABP- II protein along with selected mutants of each for the determination of the dissociation constant for retinoic acid, chemical modification and substrate protection experiments and circular dichroism studies. The determination and comparison of the retinoic acid binding site of CRABP-I and CRABP-II will provide useful information related to the overall mechanism of action of retinoic acid. In addition, this information will be extremely useful in the design of new, safer retinoids for the treatment of such diseases as cancer and skin disorders.
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