? Fibroblast growth factor homologous factors (FHFs) bear sequence and structural homology to fibroblast growth factors (FGFs), but FHFs act solely as neuronal intracellular signalling molecules. FHFs are co-factors for the assembly of the kinase module and are themselves phosphorylated in vivo. FHFs have been shown to associate with a MAP kinase scaffold protein, IB2, and with voltage-gated sodium channels in the central and peripheral nervous systems. Although the precise functions of FHFs are still poorly understood, mice bearing mutations in FHF genes are neurologically impaired. FHF1-/-FHF4 -/- double knockout mice are severely hyperactive and have very poor grip strength. Preliminary electromyography data indicate motor nerve and neuromuscular deficiencies in these animals. Continued research on FHFs shall focus on four new Specific Aims: I) The nerve conduction and neuromuscular transmission defects of FHF1-/-FHF4 -/- mice shall be determined through electrophysiological, ultrastructural, immunological, and biochemical approaches. Detected motor nerve defects shall guide analysis of higher brain centers for similar defects. Genetic interaction between mutant FHF and sodium channel genes shall be investigated. II) The IB2 gene shall be disrupted conditionally in neurons of neonatal mice. The motor phenotypes of IB2 mutant mice shall be determined and compared to those of FHF mutants, and we shall test whether reduced IB2 gene dosage potentiates FHF mutant phenotypes. III) The identity and constituents of brain microvesicles with which phosphorylated FHF associate shall be determined by conventional and affinity-based fractionation methods. IV) The structure of complexes containing FHF together with the FHF-binding segment of IB2 or sodium channels shall be determined by X-ray crystallographic methods. The structures shall guide mutagenesis to determine residues critical for interactions, determine whether IB2 and sodium channels bind FHF by a similar mechanism, and resolve why FHF and FGF folds and surfaces are so paradoxically similar. ? ?
