The growth cone at the distal tip of the extending axon is a specialized sensory apparatus which transduces extracellular signals into growth along appropriate pathways to correct synaptic targets. Its proper function is crucial to nervous system development and hence function. This proposal seeks a molecular understanding of the signal transduction mechanisms at the neuronal growth cone. Particular emphasis is focused on the action of collapsin-1, a member of the semaphorin family of proteins recently recognized to inhibit axonal extension and terminal arborization. The essential role of monomeric G proteins of the rho family and of heterotrimeric G proteins in dorsal root ganglion (DRG) growth cone signal transduction will be examined by introducing mutant activated and dominant negative proteins. The hypothesis that GAP-43 augments sensitivity of the growth cone to extracellular signals will be examined in cultured DRG neurons from mice with a targeted deletion mutation in the GAP-43 gene. A recently identified family of neuronal CRMP proteins appear to be required for collapsin-1 inhibition of growth cone function. This project will further define CRMP action by identifying proteins interacting with CRMP, exploring enzymatic activities of CRMP and comparing the properties of different CRMP family members. Neurite outgrowth and collapsin-1 sensitivity of cells overexpressing different forms of CRMP will be examined. A collapsin-alkaline-phosphatase fusion protein will be used to identify collapsin binding proteins which may serve as collapsin receptors in the neuronal growth cone. Once such receptors are identified their interaction with CRMP, GAP-43, and G proteins can be delineated. Together these experiments provide a detailed description of the molecular events which underlie the growth cone responsiveness to extracellular inhibitory signals such as collapsin-1. Knowledge of these pathways provides necessary groundwork for understanding the pathophysiology of human developmental abnormalities of the brain. The same mechanisms are likely to function during adult nervous system injury and improve function in degenerative neurologic diseases.
