Learning how axons of retinal ganglion cells grow from their origins in the retina to their targets in the brain is an important goal for understanding how the visual system develops. A number of cell adhesion molecules (CAMs) have been found in the optic fiber layer and optic nerve and are thought to be important in axon growth and guidance. A basic idea in the field has been that CAMs mediate cell-cell interactions via cell- cell binding. Over the past few years a more complex concept has evolved, i.e., that CAMs not only mediate cell adhesion but are also capable of signaling, perhaps by influencing intracellular second messenger systems. One CAM, called L1 (also referred to as NILE, Ng-CAM, 8D9 or G4) is present on axons of retinal ganglion cells. We have shown that L1 is a potent substrate for growth of retinal ganglion cell axons in vitro and that L1 binds to L1 in a homophilic manner. Using two different techniques, we have found that L1 is a strongly adhesive substrate and produces distinctive behavior by growth cones. Other labs have directly implicated L1 in regulating intracellular Ca++ and phosphorylation events, while we have identified a novel S6 kinase activity associated with L1. We have cloned and sequenced the human L1 cDNA and found that the cytoplasmic domain of L1 is highly conserved in mammals. The cytoplasmic domain of L1 in brain is 114 amino acids long and is identical in mice and humans. Using our cDNA clones, other laboratories have shown that human X-linked hydrocephalus (HSAS is due to a defect in L1 expression. Patients with this syndrome die around the time of birth or are severely retarded, have strabismus and other visual system defects. In at least one HSAS family this is doe to a mutation affecting the cytoplasmic domain of L1. L1 is an excellent CAM to study in order to understand how CAMs influence growth cone behavior by simultaneously mediating growth cone-substrate adhesion and influencing second messenger systems. The experiments in this proposal will use biochemical, immunological, molecular biological and cell biological experiments to focus on the cytoplasmic domain of L1. Site directed mutagenesis will be done on the cytoplasmic domain of L1 to define regions critical for L1-L1 homophilic binding and neurite outgrowth. We will also examine how phosphorylation of L1 is regulated and how phosphorylation of L1 influences L1 function. These experiments will provide new information about how cell adhesion modulates intracellular processes and also how changes in intracellular second messenger systems can alter cell adhesion. Finally, these experiments will provide a detailed description of how one cell adhesion molecule regulates growth cone behavior.
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