RETINOID RECEPTOR CONTROL IN CYTODIFFERENTIATION

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An initial objective of the proposed research is the elucidation of the complete genomic sequences of the retinoid-binding proteins (RBPs), cellular retinol-binding protein (cRBP), cellular retinol-binding protein (cRBP), cellular retinoic acid-binding protein (cRABP) and membrane-associated retinoic acid-binding protein (mRABP). RBPs will be purified to homogeneity and their amino-terminal ends sequenced. The amino acid sequences of these proteins and a codon frequency derived from a known, highly homologous protein will be the basis for the synthesis of several oligonucleotides. These oligonucleotides will be used as primary probes in screening complete mouse cDNA libraries; the derived cDNAs will be used to identify the complete genomic clone of cRBP, cRABP, and mRABP. Retroviruses will be constructed that contain either a normal or inverted (anti-sense) genomic sequence (from or near the amino terminal end extending 3 prime to include any primary intron) of the resolved RBP genes. These retroviruses will be infected into cell lines that have been selected for (i) their expression of RBPs cRABP+, cRBP or cRABP+/cRBP+ and (ii) their ability to respond to relatively low concentrations of retinoids retinoic acid+, or retinol+. Changes in cell characteristics (e.g., proliferation, transformation, terminal differentiation) in the presence and absence of (i) an additionally expressed RBP genomic sequence or anti-sense message; (ii) retinoids, and (iii) the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, will also be evaluated. These experiments will (i) provide the complete amono acid as well as genomic sequence for cRBP, cRABP and MRABP; (ii) establish the requirement for RBPs in the control of cytodifferentiation, (iii) allow characterization of RBP secondary structures; and (iv) provide data for the prospective development of anti-retinoid and anti-RBP compounds that may be of significant value in chemoprevention.