Our understanding of the molecular mechanisms underlying excitation in vertebrate photoreceptors improved substantially in the past decade. However, the mechanisms responsible for regulation of the transduction pathways are beginning to emerge only very recently. Understanding the regulatory pathways of transduction are no less important than understanding the transduction pathway itself because those downstream pathways maintain the integrity of photoreceptors and their function. Any defect in a regulatory pathway can cause a pathological defect in the process of visual transduction ranging in severity from a minor perturbation to a severe effect such as photoreceptor degeneration. Our hypothesis is that a series of protein phosphorylation circuits is likely to play a key role in such regulatory mechanisms and our long term goal is to understand the protein phosphorylation circuits and their roles at molecular levels. Drosophila phosrestin I (PRI) is an invertebrate counterpart of vertebrate arrestin, but differs from vertebrate arrestin because it undergoes light-induced phosphorylation in vivo. Arrestin plays a major role in the desensitization of rhodopsin by binding to its phosphorylated C-terminus. The phosphorylation of PRI is catalyzed by a calcium/calmodulin-dependent protein kinase (a CaM kinase) and is strictly regulated by cytosolic Ca2+. The activation of CaM kinase represents one bifurcation of the dual protein phosphorylation cascades downstream of polyphosphoinositide-specific phospholipase C. In order to understand the role of PRI phosphorylation, the following specific aims will be sought during the next 5 year period: 1) to determine whether the specific identity of PRI kinase is CaM kinase II, 2) to determine the phosphorylation sites of rhodopsin in vivo by mass spectrometry, 3) to determine the nature of the association of PRI with rhodopsin and the inhibitory effect on GTPase activity of different combinations of each component's phosphorylation states, and 4) to create phosrestin (both phosrestin I and II) mutants which absolutely lack PRI and/or PRII. The achievement of these aims will lead us to a better understanding of the role of protein phosphorylation cascades in the regulatory mechanisms of photoreceptor transduction.
