STUDIES OF VISUAL CYCLE PROTEINS

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The major emphasis of the proposed research is to characterize the human gene encoding the cellular retinaldehyde-binding protein (CRALBP) from retina and to identify functional domains within the three-dimensional structure of CRALBP. CRALBP could be a functional component of the visual cycle, perhaps serving as a substrate carrier and/or stereoselective agent. CRALBP carries as endogenous ligands retinoids that are only known to function in vision, namely 11-cis-retinol and 11-cis-retinaldehyde and has only been found in tissues that respond to light (retina and pineal). Appropriate attention will be given to cloning and sequence analysis of the human CRALBP gene and to identification of regulatory regions responsible for tissue-specific expression. A concerted effort will also be made to identify the domains responsible for retinoid binding and interaction with retinal pigment epithelium (RPE) retinol dehydrogenase and retinyl ester synthase. The cellular localization of the protein will be confirmed by in situ techniques, the chromosomal location of the human gene determined and clinical linkage studies pursued. The possible involvement of CRALBP in hereditary retinal diseases such as retinitis pigmentosa is of major interest. A long-range goal is to define through genetic/protein engineering studies the structural features of the protein that are responsible for its high stereoselectivity and low photosensitivity. Regulatory regions of the gene will be characterized by primary RPE and Muller cell culture transfection CAT assays. CRALBP mRNA levels will be measured in RPE and Muller cell culture following treatment with various steroid hormones, growth factors and retinoids. The whole protein will be chemically synthesized in a limited number of peptides (16-30 residues/peptide) by automatic solid phase methods and anti-CRALBP peptide antibodies raised in rabbits. Functional domains will be probed by screening for function-blocking antibodies and peptides and by limited proteolysis. The combined results from the proposed research will provide a precise molecular framework in which questions concerning the normal function of CRALBP and the visual disorders it may be associated with can be answered in specific terms.