The Multifunctional Protein Gephyrin

描述(申请人提供)：突触代表中枢神经系统中神经元之间的一种特殊结构。配体门控离子通道超家族神经递质受体负责突触部位兴奋性和抑制性信号的快速传递，它们在突触后部位的定位对突触的有效传递至关重要。突触后部位以膜下细胞骨架元素密集堆积为特征。哺乳动物蛋白Ge对抑制甘氨酸和GABAA受体的聚集起着至关重要的作用。通过同时与受体的13个亚基和微管蛋白结合，吉卜林将甘氨酸受体锚定在细胞骨架上。此外，Geporrin还与其他蛋白质相互作用，这些蛋白质可能在突触后密度的组装中发挥重要作用，包括胶原蛋白、RAFT1、Profilin和GABARAP。 Gen被认为在突触后膜下形成一个六角形的支架，为细胞骨架的受体和元件提供结合位置。这项提案的总体目标是评估和扩展这种脚手架模式。一个潜在的假设是，与突触后膜组织相关的功能分布在其整个初级序列中，而不是像通常认为的那样，仅限于连接区。这一策略将允许地卟啉同时参与多个结合相互作用，从而调节其几个结合伙伴的活动。这一假设的第二个假设是，配对蛋白的结合影响了Gen的低聚状态，从而影响了它在突触后膜下形成六角形支架的能力。 为了研究支架模型，本论文将利用生化和结晶学技术对地卟啉及其配合物进行分析，以了解其功能的多样性。具体地说，该提案将确定吉普林中负责识别其结合伙伴的区域。蛋白质-蛋白质复合体的强度及其低聚状态将通过生物物理技术进行分析。这些研究将通过对全长Geporrin、其E结构域以及由该蛋白质形成的各种蛋白质-蛋白质复合体的晶体结构分析来补充。这些实验将促进人们对地理卟啉在组织抑制性突触的突触后膜中的多重功能的理解，并将检验和扩展地理卟啉的支架模型。

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.