The aim of the research proposed in this application is to gain insight into the molecular mechanisms of signal transduction in the visual system. It is expected that these studies will contribute to a better understanding of the relationship between receptor structure, function, and information processing in biological systems. The genes encoding four Drosophila opsins have been isolated and characterized (Rh1-4). Each of these is expressed in distinct classes of photoreceptor cells and displays characteristic patterns of sensitivity to light. The Rh1 opsin is a blue sensitive pigment (lambda/max 480nm) which is expressed in the six outer photoreceptor cells (R1-R6). Rh2 displays light sensitivity with lambda/max 420nm and is expressed in the occelli. Rh3 and Rh4 are UV sensitive and are expressed in non-overlapping sets of R7 cells. In previous studies, we generated a series of chimeric Drosophila opsin genes, in order to identify regions of the opsin protein that are involved in regulating rhodopsin sensitivity and metarhodopsin absorption. These chimeric proteins were derived from regions of the blue and violet sensitive pigments. The chimeric genes were expressed in the R1-R6 photoreceptor cells of ninaE mutant animals which lack the wild type Rh1 gene product. In these flies, the chimeric opsins are the only photopigments expressed in the R1-R6 photoreceptor cells. We examined the spectral sensitivity of animals expressing these modified pigments in vivo using physiological techniques, and studied the absorption of the activated form of the pigment (metarhodopsin) by in vivo microspectrophotometry. We found that multiple regions of the opsin protein are involved in regulating rhodopsin spectral sensitivity and that the native and activated forms of the pigment can be tuned independently. In the present application we propose to 1) identify regions of the opsin protein that are responsible for differences in the spectral sensitivity of the Drosophila Rh1 and Rh2 rhodopsins. 2) Identify residues in the second transmembrane segment of the opsin protein that are responsible for differences in the absorption of the activated form (metarhodopsin) of the Drosophila Rh1 and Rh2 rhodopsins. 3) Identify additional sites within the opsin protein that play an important role in rhodopsin sensitivity and function. 4) Isolate and characterize the genes encoding novel photopigments found in Drosophila and other invertebrates. 5) Generate and characterize transgenic flies that ectopically express opsins from other invertebrate and vertebrate organisms. These studies will provide insight into the mechanisms underlying the regulation of rhodopsin spectral sensitivity and the relationship between rhodopsin structure and the process of photoactivation. These issues are central to the study of vision, and will provide a unique model system for understanding how G-protein coupled receptors function.
