Elucidation of molecular signaling mechanism of axonal guidance

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Collapsin response mediator proteins (CRMPs) have been implicated in signaling of axonal guidance, including semaphorins. I have previously identified a novel CRMP-associated protein, designated CRAM for CRMP-Associated Molecule that belongs to the unc-33 gene family. The deduced amino acid sequence reveals that the CRAM gene encodes a protein of 563 amino acids and shows 57 % identity with dihydropyrimidinase (DHPase) and 50-51 % identity with CRMPs. A phylogenetic tree analysis indicates that CRAM sequence shows the greatest similarity with DHPase and divergence from the four CRMPs, indicating that CRAM is more closely related to DHPase than to the four CRMPs. The expression of CRAM is remarkable for brain, and especially high in fetal and neonatal, but decreases to very low levels in adult brain. Thus, CRAM is a unique new member of this gene family and may play a role in axonal guidance signaling distinct from other four CRMPs. Furthermore I have recently reported that CRAM was associated with several cellular proteins including tyrosine kinase Fes/Fps and mitochondrial septin.In this study, I have examined the distribution and function of CRAM in developing neurons. Immunohistochemical analysis showed accumulation of CRAMin the filopodia of growth cones. Experiments using cytochalasin D indicated that filopodial localization of CRAM was independent of filamentous actin. Overexpression of CRAM in neuronal cells significantly promoted filopodial growth and led to the formation of supernumerary growth cones, which acquired resistance to semaphorin-3A stimulation. Finally, knockdown of CRAM by using RNA interference blocked filopodial formation and revealed an aberrant morphology of growth cones. I propose that CRAM regulates filopodial dynamics and growth cone development, thereby restricting the response of growth cone to repulsive guidance cues.