NEURONAL FUNCTIONS OF FHFS

描述(由申请人提供)： 成纤维细胞生长因子同源因子(FHFs)与成纤维细胞生长因子(FGFs)具有序列和结构上的同源性，但FHFs仅作为神经细胞内信号分子发挥作用。FHFs是组装激酶模块的辅助因子，它们本身在体内被磷酸化。FHFs已被证明与MAP激酶支架蛋白IB2以及中枢和外周神经系统中的电压门控钠通道有关。尽管FHF的确切功能仍然知之甚少，但携带FHF基因突变的小鼠在神经学上是受损的。FHF1-/-FHF4-/-双基因敲除小鼠严重过度活跃，握力非常差。初步的肌电数据显示这些动物有运动神经和神经肌肉缺陷。FHF1-/-FHF4-/-小鼠的神经传导和神经肌肉传递缺陷将通过电生理、超微结构、免疫学和生化方法确定。检测到的运动神经缺陷应指导对类似缺陷的高级大脑中枢的分析。应研究突变的FHF和钠通道基因之间的遗传交互作用。Ii)新生小鼠神经元中的IB2基因应被有条件地破坏。我们将测定IB2突变小鼠的运动表型，并与FHF突变小鼠的运动表型进行比较，我们将测试IB2基因剂量减少是否会增强FHF突变表型。3)与磷酸化FHF结合的脑微囊的特性和成分应通过常规和基于亲和力的分级方法确定。IV)含有FHF和IB2或钠通道的FHF结合片段的络合物的结构应用X射线结晶学方法确定。这些结构将指导突变以确定相互作用的关键残基，确定IB2和钠通道是否通过类似的机制结合FHF，并解决为什么FHF和FGF的折叠和表面如此矛盾地相似。

DESCRIPTION (provided by applicant): 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.