This research project will investigate the role of EphB receptor signaling in dendritic spine development. Understanding the molecular basis of dendritic spine morphogenesis is fundamentally important to a variety of inherited developmental disorders associated with mental retardation and autism, including Rett Syndrome and FragileX Syndrome. Patients with these disorders exhibit malformation of dendritic spines. These abnormalities result in synaptic dysfunctions, mental retardation and autism. The molecular mechanisms of dendritic spine abnormalities are not well described and require further investigation. Recently I made an important discovery that spine morphogenesis is controlled by the EphB-type receptor tyrosine kinases (Ethell et al., Neuron, 2001). I showed that expression of kinase-inactive EphB2, which prevents activation of EphB-type receptors in a dominant-negative fashion, blocked spine formation in cultured hippocampal neurons, the dendritic protrusions remained long, thin filopodia, as seen in patients with mental retardation and autism. I hypothesize that EphrinB (ligand)-induced activation of EphB receptors control dendritic spine formation. Preliminary results support this hypothesis and shows that clustered EphrinB2-Fc promotes dendritic spine morphogenesis. In Specific Aim 1, I will conduct experiments with knock out mice in which expression of one or multiple EphB receptors is disrupted to find which of the EphB receptors is responsible for the EphrinB2-induced spine formation. I propose to investigate two possible mechanisms through which EphB receptors may trigger dendritic spine mophogenesis: 1) recruitment of signaling molecules to synaptic membranes; 2) tyrosine phosphorylation of key molecules at postsynaptic sites. In Specific Aim 2, I will identify signaling molecules that may link the signaling of the EphB receptors to its effect on spine formation, by conducting mass-spectrometry analysis of the proteins recruited by EphB2 to dendritic spines upon its activation with EphrinB2-Fc. Preliminary results suggest that RhoGTPases may be responsible for the EphrinB/EphB receptor-mediated dendritic spine formation. In Specific Aim 3, I propose to investigate the molecular mechanism of EphB-mediated regulation of RhoGTPases in dendritic spines and its correlation with EphrinB-induced spine formation. In Specific Aim 4, I will also investigate role of cell adhesion molecules in EphrinB-mediated formation and stabilization of dendritic spines.