The regulation of tyrosine kinase phosphorylation is critical for the growth and guidance of axons. Evidence suggests that the CAM-like receptor-tyrosine phosphatases (RPTPs) play key roles in the signaling processes underlying axon growth. This project tests the hypothesis that RPTPs, expressed on the surface of developing neurons regulate axon growth during embryogenesis, through the binding of specific ligands to their extracellular domains. The focus here is on 2 specific RPTPs -- CRYP-2 and PTP-deltadelta -- that are excellent candidates for involvement in the regulation of axon growth. Evidence implicating these two RPTPs includes structure, expression pattern, and preliminary analysis of function in vitro. The major questions concerning these RPTPs are: What are the regulatory ligands for CRYP-2 and PTP-delta?, and How are CRYP-2 and PTP-delta involved in axon growth? These questions will be addressed through correlation of expression of ligands for CRYP-2 and PTP-delta with expression of the RPTPs themselves, characterization of these ligands, and examination of the function of CRYP-2 and PTP-delta in axon growth, both in vitro and in vivo. There are three specific aims. First, potential regulatory ligands for the RPTPs will be localized, and identified through immunoprecipitation, affinity chromatography and expression cloning. Identification of ligands will allow key biochemical experiments on adhesive and signaling functions of these RPTPs. Second, the extracellular domains of the RPTPs will be used as regulators of axon growth and guidance in vitro, in an analysis of the mechanisms involved in this regulation. These results will provide detailed mechanistic evidence concerning how these RPTPs function. Finally, two different in vivo assay systems (retroviral expression of mutants and gene knockout) will be employed to assess the role of these RPTPs in neuronal development during embryogenesis. These assays will provide direct tests of the hypothesis that the specific PTPs under study are functionally linked to axon growth and guidance in vivo. The proposed experiments will move the field closer to a molecular understanding of the regulation of vertebrate axon growth. This understanding is critical for rational approaches to developmental disorders of the nervous system, as well as the problem of spinal cord regeneration.
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