The overall goal is to elucidate the operation, within intact retinal pigment epithelium (RPE), of retinoid processing reactions that mediate 11- cis retinal formation and thus support rhodopsin regeneration in the rods. Results obtained from the toad (B. marinus) """"""""RPE-eyecup"""""""" (posterior eyecup devoid of neural retina) show that interphotoreceptor retinoid-binding protein (IRBP), a soluble glycoprotein of the interphotoreceptor matrix, markedly stimulates the RPE's synthesis of 11-cis retinal from all-trans precursor, as well as the release of this cis retinal to the apical extracellular medium. The experiments are aimed at determining how IRBP exerts these effects on RPE retinoid processing, and how progressive rod degenerative disease affects the status of the processing reactions. Under defined conditions of treatment with IRBP, extracellular retinoid, and illumination pre-bleaching of the native eyecup), the fate of (3H) retinoid incorporated into the RPE will be examined in toad RPE-eyecups.
Specific aims are to determine the activities of 11-cis retinyl ester as a precursor of 11-cis retinal or (by back isomerization) trans ester, and whether such reactions are IRBP-dependent. The """"""""VPP"""""""" transgenic mouse, a well-studied model of slowly progressing photoreceptor degeneration, will be studied model of slowly progressing photoreceptor degeneration, will be used in experiments to analyze bleach-induced changes in retinoid levels within the retina and RPE in vivo. A key aim is to determine whether retinoid processing becomes impaired at early times in this opsin-linked degenerative disease. In further experiments using toad RPE-eyecup, recombinant IRBP polypeptides of defined sequence (to be provided by our collaborators) will be tested for activity in promoting 11-cis retinal release from, and all-trans retinol delivery to, the RPE. The observed retinoid transfer activities of test polypeptides will be interpreted in relation to their (native and mutant) amino acid sequences, with the aim of elucidating the basis of native IRBP's action at the RPE apical membrane.
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