The Multifunctional Protein Gephyrin

描述（由申请人提供）：突触代表中枢神经系统中神经元之间通信的专门结构。神经递质受体的配体门控离子通道超家族的成员负责兴奋和抑制信号在突触位点的快速传递，并且它们在突触后位点的定位对于有效的突触传递至关重要。突触后部位的特征是膜下细胞骨架成分的密集堆积。哺乳动物蛋白质桥蛋白对于抑制性甘氨酸和GABAA受体的聚集是至关重要的。桥蛋白通过同时结合受体的13-亚基和微管蛋白将甘氨酸受体锚定到细胞骨架。此外，桥蛋白与其他蛋白质相互作用，可能在突触后密度的组装中发挥重要作用，包括collybistin，RAFT 1，profilin和GABARAP。 桥卟啉被认为在突触后膜下形成六边形支架，为受体和细胞骨架元件提供结合位点。本提案的总体目标是评估和扩展这种脚手架模型。一个潜在的假设是，桥蛋白的功能有关的组织突触后膜分布在整个其主要序列，而不仅仅是局限于连接区已普遍假设。这种策略将允许桥蛋白同时参与多种结合相互作用，从而调节其几种结合伴侣的活性。该提议的第二个假设是，伴侣蛋白的结合影响桥蛋白的寡聚状态，从而影响其在突触后膜下形成六边形支架的能力。 为了研究支架模型，桥蛋白及其复合物将通过生物化学和晶体学技术进行分析，以了解其功能多样性。具体而言，该提案将确定负责识别其结合伙伴的桥蛋白区域。蛋白质-蛋白质复合物的强度及其寡聚状态将通过生物物理技术进行分析。这些研究将通过全长桥蛋白、其E结构域和由该蛋白形成的各种蛋白质-蛋白质复合物的晶体结构分析来补充。这些实验将推进桥蛋白在抑制性突触组织突触后膜的多种功能的理解，并将测试和扩展桥蛋白的支架模型。

DESCRIPTION (provided by applicant): The synapse represents a specialized structure for communication between neurons in the central nervous systems. Members of the ligand-gated ion channel superfamily of neurotransmitter receptors are responsible for rapid transmission of excitatory and inhibitory signals at synaptic sites and their localization at postsynaptic sites is vital for efficient synaptic transmission. The postsynaptic sites are characterized by dense accumulations of submembranous cytoskeletal elements. The mammalian protein gephyrin is crucial for the clustering of inhibitory glycine and GABAA receptors. Gephyrin anchors glycine receptors to the cytoskeleton through simultaneous binding to the 13-subunit of the receptor and tubulin. In addition, gephyrin interacts with other proteins presumably playing important roles in the assembly of postsynaptic densities, including collybistin, RAFT1, profilin and GABARAP. Gephyrin has been postulated to form a hexagonal scaffold underneath the postsynaptic membrane, which provides binding sites for the receptors and elements of the cytoskeleton. The overall goal of this proposal is to evaluate and expand this scaffolding model. One underlying hypothesis is that the functions of gephyrin pertaining to the organization of the postsynaptic membrane are distributed throughout its primary sequence and are not only confined to the linker region as has been generally assumed. This strategy would allow gephyrin to simultaneously engage in multiple binding interactions, thus modulating the activities of several of its binding partners. A second hypothesis of this proposal is that binding of the partner proteins influences the oligomeric state of gephyrin and consequently its ability to form the hexagonal scaffold underneath the postsynaptic membrane. In order to investigate the scaffolding model, gephyrin as well as its complexes will be analyzed by biochemical and crystallographic techniques in order to understand its functional diversity. Specifically, the proposal will identify regions in gephyrin responsible for recognition of its binding partners. The strengths of the protein-protein complexes and their oligomeric states will be analyzed by biophysical techniques. These studies will be complemented by crystal structure analyses of full-length gephyrin, its E-domain and the various protein-protein complexes formed by this protein. These experiments will advance the understanding of the multiple functions of gephyrin in organizing the postsynaptic membrane at inhibitory synapses and will test and extend the scaffolding model of gephyrin.