Studies from our laboratory over the past two decades have shown that the retina and rod outer segments (ROS) have an active phosphoinositide (P1) metabolism (called the P1 Cycle) that generates several lipid second messengers. During the last year, we discovered that the insulin receptor -subunit (IR) is tyrosine phosphorylated in vivo and in vitro in response to light and directly activates PT 3-kinase, which forms PI-3,4,5-P3 and other D3-phosphorylated PIs in ROS. In other tissues, these second messengers have been shown to affect a large number of intracellular events, including secretion, cell survival, membrane trafficking, and actin polymerization, all of which are important in the retina. It is our hypothesis that light-stimulation of this reaction in photoreceptor cell outer segments affects important photoreceptor cell function(s) not related to visual transduction. The long-term goal of our research is to elucidate the physiological role of these lipid second messengers in photoreceptor cells. To this end, we will determine the mechanism of activation of the enzymes that synthesize these messengers and identify their downstream effectors. Expression of GST-fusion proteins containing the JR motif will be used in pull-down assays to obtain proteins that can be identified on Western Blots, by 2D gel electrophoresis followed by in-gel digestion and mass spectrometry of the peptide fragments, or by protein sequencing followed by cloning and sequencing the gene. Once these proteins have been identified, we will attempt to determine their role in the light-driven reactions involving the insulin receptor and the P1 Cycle. We will use new gene knockout technology now available in our laboratory to selectively eliminate the gene products of several enzymes of the P1 Cycle in photoreceptor cells and determine the effect on photoreceptor structure and function.
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