The Multifunctional Protein Gephyrin

多功能蛋白Gephyrin

• 批准号: 6987892
• 负责人: HERMANN SCHINDELIN
• 金额: $ 23.79万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6987892
• 关键词: G protein; SDS polyacrylamide gel electrophoresis; actin binding protein; analytical ultracentrifugation; binding sites; glycine receptors; ligands; protein binding; protein protein interaction; protein structure function; synapses; tubulin; yeast two hybrid system

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.

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特殊结构。神经递质受体配体门控离子通道超家族的成员负责突触位点兴奋性和抑制性信号的快速传递，它们在突触后位点的定位对于有效的突触传递至关重要。突触后位点的特征是膜下细胞骨架元件的密集积累。哺乳动物蛋白 gephyrin 对于抑制性甘氨酸和 GABAA 受体的聚集至关重要。 Gephyrin 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上。此外，gephyrin 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 Collybistin、RAFT1、profilin 和 GABARAP。 Gephyrin 被认为在突触后膜下方形成六角形支架，为受体和细胞骨架元件提供结合位点。该提案的总体目标是评估和扩展这个脚手架模型。一个潜在的假设是，与突触后膜组织有关的 gephyrin 功能分布在其整个一级序列中，而不仅限于通常假设的连接区。这种策略将允许 gephyrin 同时参与多种结合相互作用，从而调节其几个结合伙伴的活性。该提议的第二个假设是，伴侣蛋白的结合影响 gephyrin 的寡聚状态，从而影响其在突触后膜下方形成六角形支架的能力。 为了研究支架模型，将通过生化和晶体学技术对 gephyrin 及其复合物进行分析，以了解其功能多样性。具体来说，该提案将确定 gephyrin 中负责识别其结合伙伴的区域。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。这些研究将得到全长 gephyrin、其 E 结构域以及由该蛋白质形成的各种蛋白质-蛋白质复合物的晶体结构分析的补充。这些实验将增进对 gephyrin 在组织抑制性突触突触后膜方面的多种功能的理解，并将测试和扩展 gephyrin 的支架模型。