MOLECULAR MECHANISMS UNDERLYING GAP-43 FUNCTION

描述：大量证据表明神经细胞生长 相关蛋白GAP-43在神经可塑性调控中的作用 既有发展，也有学习。然而，其分子机制 GAP-43控制这些功能尚不清楚。本提案使用 GAP-43功能结构域的分析及其研究 与细胞内膜调节蛋白Rabaptin-5的相互作用 Fusion，以描绘参与GAP-43参与的分子事件 神经突起生长和神经递质释放。首先，假设 GAP-43的特定结构域可以链接到特定的信号通路和 它在不同的神经递质释放和释放过程中的作用 将对轴突生长进行测试。破坏功能的GAP-43突变体 该蛋白的结构域将在PC12细胞和神经元中表达 GAP-43。这些突变体与钙调蛋白、蛋白激酶的相互作用 C，并对GTP结合蛋白G0进行评估，随后 这些突变对神经递质释放和突起发育的影响 将会被量化。需要检验的第二个假设是，已知的 GAP-43与钙调蛋白和蛋白激酶C的相互作用与其 突触前终末与Rabaptin-5的相互作用。域映射， 共同定位研究和定量功能分析将澄清 GAP-43与Rabaptin-5结合的功能作用 涉及这些分子的胞膜融合事件是否包括 神经递质释放和轴突生长之间的分子联系。 从拟议研究中获得的数据将开始提供统一的 GAP-43已知多种功能的分子机制 参与进来。

DESCRIPTION: A large body of evidence implicates the neuronal growth associated protein GAP-43 in the modulation of neuronal plasticity during both development and learning. However, the molecular mechanisms by which GAP-43 controls these functions are not known. The present proposal uses analyses of functional domains of GAP-43, coupled with studies of its interaction with rabaptin-5, a protein that mediates endocytic membrane fusion, to delineate the molecular events underlying GAP-43 participation in neurite outgrowth ad neurotransmitter release. First, the hypothesis that specific domains of GAP-43 can be linked to specific signaling pathways and to its roles in the distinct processes of neurotransmitter release and neurite outgrowth will be tested. GAP-43 mutants that disrupt functional domains of the protein will be expressed in PC12 cells and neurons lacking GAP-43. The interactions of these mutants with calmodulin, protein kinase C, and the GTP-binding protein G0 will be evaluated, following which the effects of these mutations on neurotransmitter release and process outgrowth will be quantified. The second hypothesis to be tested is that the known interactions of GAP-43 with calmodulin and protein kinase C are tied to its interactions with rabaptin-5 in the presynaptic terminal. Domain mapping, co-localization studies, and quantitative functional assays will clarify the functional role of the binding of GAP-43 to rabaptin-5, and demonstrate whether endocytic membrane fusion events involving these molecules comprise a molecular link between neurotransmitter release and neurite outgrowth. The data obtained from the proposed studies will begin to provide unifying molecular mechanisms for the diverse functions in which GAP-43 is known to participate.