Light stimulates the hydrolysis of a phospholipid, phosphatidylinositol 4,5-bisphosphate (PlP2), in vertebrate and invertebrate retinas, which generates two intracellular second messengers. One, 1,4,5-inositol trisphosphate (1,4,5-lP3), a water soluble molecule, interacts with specific intracellular receptors and causes release of bound calcium. The other, 1,2-diacylglycerol (1,2-DG), a lipid molecule, activates protein kinase C (PKC). Five types of PLCs have been described that are specific for the hydrolysis of PIP2. In bovine ROS, PLC activity is activated by arrestin (aka 48K protein and S-antigen). Our long-term goal is to understand the mechanism of generation and the role of PIP2-derived second messengers in the vertebrate retina.
The specific aims for the next five years are: 1) to identify the PLC subtypes in the retina by probing with a variety of PLC-specific antibodies, using immunocytochemistry and immunoblots. 2) to characterize the PLCs in the retina and ROS. PLCs will be purified from ROS and the mechanism of activation by arrestin will be determined. Antibodies will be generated against purified PLCs and used to screen expression libraries so that the PLCs can be cloned and their genes sequenced. 3) to localize and identify the inositol phosphate receptors in the retina, using subcellular fractionation and immunocytochemistry. 4) to establish a reconstituted system for study of light-stimulated PIP2 hydrolysis. These studies will utilize biochemical, morphological, immunological, and molecular biological techniques. This expertise is available in the PI's laboratory; however, collaborations with experts in specific areas have been arranged, also. The result of these studies will be more precise information about the role of PIP2 hydrolysis in the retina and rod outer segments. Since this reaction is involved in control of calcium levels in cells, our hypothesis is that it may be involved in adaptation of photoreceptor cells. To date, there are no known genetic defects in the vertebrate visual system involving enzymes of the PIP2 cascade. However, abnormalities in both PLC and PKC in Drosophila eyes are associated with aberrant retinal function and/or retinal degeneration.
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