The long term goal of this project is to understand the molecular events underlying the function of visual photoreceptor cells, especially the processes leading to visual sensation, i.e., excitation, transduction, adaptation, and restoration of sensitivity. In order to achieve this goal, the fruit fly, Drosophila melanogaster, will be used as a major experimental system in this proposal. The use of Drosophila as a model system to study general rules underlying the physiological functions of higher vertebrates including humans is now widely accepted. The rationale of this approach is based on the fact that Drosophila shares many proteins exhibiting a high degree of similarity with those of vertebrates. Moreover, Drosophila is one of the few eucaryotic systems in which the most advanced genetic and molecular biological techniques can be applied. As a short term goal, this project aims to elucidate the role of protein phosphorylation in visual photoreceptor function. It is well established that phosphotidylinositol-specific phospholipase (PI- PLC) plays a key role in second messenger systems involving calcium- and protein kinase C-mediated processes in a wide variety of cell types. The photoreceptor transduction of dipteran flies is likely to involve the activation of PI-PLC. Based on the results of our previous project, it is proposed that the light-induced phosphorylation of the 49K protein of Drosophila photoreceptor is regulated by Ca2+, the concentration of which is likely to be regulated by PI-PLC. The goals of the project are: 1) The gene structure of the 49K protein, including the DNA sequence of cDNA encoding the 49K protein, will be determined. 2) The 49K protein will be purified under mild conditions in order to examine possible protein kinase activity and possible Ca2+ binding. 3) Partial amino acid sequences of CNBr-cleaved 49K protein fragments will be determined and the fragment to which the phosphate group is attached will be determined. 4) Spatial and temporal (developmental) expression of the 49K protein will be examined by use of antibody and RNA probes both at light and electron microscope levels. 5) Mutations at the 49K protein locus will be created in order to examine the phenotypic expression of the 49K gene when it is defective.
