(Applicant 's abstract) Tyrosine-specific protein kinases are abundant in synaptic layers of the vertebrate neural retina but their function remains a central unanswered question in vision research. We recently made the important discovery that tyrosine (Tyr) phosphorylation of specific proteins (106, 78, 40 Kd and others) associated with terminals of retinal neurons and surrounding Muller glial processes in the chick neural retina is dramatically stimulated in response to illumination. The response is rapid, nontransient, and readily reversible. Our immediate goal is to define the proximal components of the pathway of signal transduction mediated by Tyr kinases in response to light (the neurotransmitter signals, the Tyr kinases activated by light, and the substrates of kinase action) with the long range goal of elucidating their function in the photic response. Our working hypothesis is that Tyr dephosphorylation of substrates is related to neurotransmitter signalling or reuptake in retinal neurons or glia. We will apply a new tool, phosphotyrosine (PTyr) antibodies to analyze the PTyr response in the chick retina by immunocytochemical, biochemical, and molecular biological techniques. We will (1) determine the optimal illumination conditions pathways, (2) use immunoelectron microscopy to localize the subcellular sites of the substrates and to test the postulate that the PTyr response is related to coated vesicle dynamics involved in membrane recycling, (3) identify specific neurotransmitters as signals for Tyr dephosphorylation or phosphorylation in the outer and inner plexiform layers, (4) generate monolconal antibodies to study the structure and function of retinal substrates, and (5) identify novel Tyr kinases responsible for light-dependent phosphorylation of substrates in the retina by PTyr antibody screening of a retinal cDNA library. This research investigates an unexplored areas of protein modification in the retina, which will contribute to our understanding of normal retinal function and vision.
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