The long term goal of our laboratory is to understand how neurons make the decision to change from one functional state to another, and how this decision is expressed as an alteration of their molecular configurations. Toward this end, we propose to investigate three problems concerned with the role of axonally transported proteins in the maintenance and changes in neuronal state. First, we will determine the amino acid sequence of a major rapidly axonally transported protein of unknown identity, with the goal of characterizing it sufficiently to understand its function. These experiments will lead to a better understanding of the function of this class of axonally transported proteins, which is characteristic of both developing and mature axons. Second, we propose to characterize further the rapidly axonally transported protein GAP-43, whose synthesis is associated with axon growth and whose phosphorylation is associated with synaptic plasticity. We will precisely define the sites of phosphorylation on the GAP-43 protein, and determine how the occupancy of these sites differs in different cellular compartments (cell bodies, growth cones and synapses) and under different physiological conditions. In addition, we will establish a physical map of GAP-43 by first determining its shape by electron microscopy, and then determining the location of certain amino acid sequences (defined by monoclonal antibodies) and certain functional domains (e.g., the calmodulin-binding domain) within the structure of GAP- 43. Furthermore, we will determine whether an unusual GAP-43 cDNA corresponds to an alternatively spliced mRNA, which would suggest that the synthesis of GAP-43 can be regulated in part by information that is prerequisite for understanding the role of GAP-43 in axon growth and synaptic plasticity. Third, we will determine the genetic basis of a naturally occurring polymorphism in the rabbit gene that specifies the neurofilament protein H, which participates in the crosslinks between neurofilaments in mature axons. The results will reveal how the structure of the rabbit H protein has been altered in the course of evolution and may suggest mechanism for the evolution of the H gene.
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