The significance of the neuronal growth cone in the formation of specific, connections during the development of the nervous system has been well established. This proposal describes experiments designed to investigate how one of the major components of growth cone membranes, the growth-associated 'protein GAP-43, is regulated in the growth cone, with the eventual goal of understanding how the growth cone is able to respond to extracellular signals and transduce them into intracellular events. The protein GAP-43 is regulated in two ways; its amount is regulated so that it is induced in neurons that are growing axons during development or regeneration, but interestingly it cannot be re-induced in the injured axons of neurons which are unable to regenerate, implying that it may perform a function intimately connected with axon growth. Second, its phosphorylation is regulated by other kinases and by autophosphorylation. These experiments use both primary neuronal tissue culture and a preparation of isolated growth cones to ask the following questions. Firstly, is GAP-43 phosphorylated by more than one kinase, and is that phosphorylation restricted to particular areas of the neuron? Second what are the extracellular signals that stimulate these kinases and does phosphorylation of GAP-43 regulate its subcellular distribution? Finally, GAP-43 is able to autophosphorylate; does this ability regulate its subcellular distribution or association with other proteins? or is GAP-43 able to phosphorylate other growth cone proteins, and so regulate their behavior. GAP-43 is associated with membranes even though it is not hydrophobic. These experiments will the nature of GAP-43 association with membranes and also the nature of a small group of three membrane proteins with which it is closely associated. Finally GAP-43 is a component of membrane that copurifies with actin microfilaments. The nature of this interaction and its regulation will be investigated. Answers to these questions will increase our understanding of the role of GAP- 43 in the growth cone, but also, because the experiments will develop a model system, will enable us to investigate more general questions about the role of signal transduction in the growth cone, with the eventual goal of using this knowledge to design strategies which circumvent the failure of some neurons (such as mammalian CNS) to regenerate successfully.
