STRUCTURE/FUNCTION ANALYSIS OF RANBP2 IN THE NEURORETINA

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The vertebrate retina is an excellent model system to study basic neurobiological processes because it is a well defined stratified neuronal tissue that is highly amenable to a wide variety of experimental manipulations. Moreover, the retina has closely related classes of neuronal cells, the cone and rod photoreceptors, which are morphologically and functionally alike, but otherwise distinct in many, yet unknown, molecular components and mechanisms governing their function. Photoreceptors are highly polarized and compartmentalized cells. They represent the epitome of a cell adapted to capture and process photon stimuli. Although a great deal is known about the role of many photoreceptor components in the light- activated cascade, very little is understood on how these components are correctly processed and sorted, in a vectorial fashion, to the outer segment subcellular compartment of photoreceptors. In particular, the molecular mechanisms underlying the processing, sorting and functional expression of the seven-transmembrane light-receptors, opsins, remains to be dissected. Indeed, this information is lacking for all seven-transmembrane receptors. Opsin constitutes about 90% of the total membrane protein in the outer segments of photoreceptors. These cells likely express a highly efficient biogenic machinery to functionally express the vast amounts of opsin that are continuously produced by these cells. Thus, photoreceptors are also an excellent model systems to study the biogenesis of seven-transmembrane receptors. Our long term goal is to identify the molecular components and mechanisms underlying the biological processes of opsin biogenesis in photoreceptor cells. This is also important if one wants to understand the molecular pathogenesis of inherited retinopathies caused by mutations in opsins. Recently, we have molecularly identified and partially characterized a novel component of the photoreceptor machinery, a cyclophilin-related protein, that selectively chaperones and enhances the functional production of long wave length visual receptors. This novel component is identical to the multi-domain protein, RanBP2. The overall focus of this proposal is to molecularly dissect the role of RanBP2 and its modulating molecular assemblies in phtoreceptor function and, in particular, in biogenesis of color opsins. To this end, we will undertake a multi-disciplinary approach of molecular, biochemical, genetic and cell biology techniques. This proposal will address these specific questions. 1. What are the molecular determinants in red opsin important for the recognition of RBD4 ane CY-PPlase domains of RanBP2? 2. What is the function of the remaining primary structural modules of RanBP2? 3. What are the effects of RanBP2 in the physiological production of red- green pigment in photoreceptors?