The long-term goal of this project is to understand the mechanisms whereby neurotrophic agents, paradigmatically represented by nerve growth factor (NGF), promote survival, differentiation, plasticity and repair of neurons. Such insight is relevant to uncovering the causes and devising ameliorative or preventive treatments for developmental and degenerative disorders affecting neurons and for formulating means to hasten or promote nerve regeneration. This application specifically focuses on dissecting functional domains of the newly discovered trk NGF receptor and on establishing causal relationships between various trk- activated intracellular signalling pathways and functional responses to NGF such as neurite outgrowth and maintenance of survival. The proposed experiments will also aim at understanding how trk receptors are responsible for generating both diversity and specificity of NGF responses. To achieve these ends, selectable mammalian expression vectors will be constructed that carry cDNAs encoding trk NGF receptors that are specifically mutated in a variety of intracellular domains that our sequence analyses suggest to be associated with particular intracellular signalling functions. These will be transfected into a neuronal cell line (PC12nnr5) derived in this laboratory that is specifically deficient in expression of normal trk NGF receptors and consequently in the capacity to respond to NGF or to normally bind or internalize this factor. Transfection of non-mutated normal trk into these cells endows them with the ability to react to NGF by expressing a large number of responses found in other NGF target cells. PC12nnr lines transfected with various mutant trks will be selected and expanded and then screened for activation by NGF of a number of specific signalling pathways as well as for a variety of subsequent transcription- dependent and -independent NGF responses. Analysis of the results of these experiments should reveal: 1) How diversity of NGF actions is generated by the presence on trk NGF receptors of distinct intracellular domains, each of which is responsible for activation of separate signalling pathways. 2) The identities of the intracellular domains of trk NGF receptors responsible for activating specific intracellular signalling pathways. 3) The functional consequences that occur when mutant trk receptors lose the capacity to activate specific intracellular signalling pathways. 4) The causal relationship between specific signalling pathways and expression of subsequent responses to NGF. 5) Particular intracellular domains of trk that endow it with signalling properties responsible for the specificity of NGF actions. 6) Intracellular domains of trk responsible for high affinity NGF binding and internalization.
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