The pentameric glycine receptor (GlyR), comprising the α1 and β subunits, is a major inhibitory ionotropic receptor in brainstem and spinal cord. GlyRs interact with gephyrin (GPHN), a scaffold protein that anchors the GlyR in the plasma membrane and enables it to form clusters in glycinergic postsynapses. Using an interaction proteomics approach, evidence of the ArfGEFs IQ motif and Sec7 domain 3 (IQSEC3) and IQ motif and Sec7 domain 2 (IQSEC2) as two novel synaptic proteins interacting with GlyR complexes is provided. When the affinity-isolated GlyR complexes are fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1β-GPHN appears as the most abundant complex with a molecular weight of ≈1 MDa, and GlyR α1β-GPHN-IQSEC3 as a minor protein complex of ≈1.2 MDa. A third GlyR α1β-GPHN-IQSEC2 complex exists at the lowest amount with a mass similar to the IQSEC3 containing complex. Using yeast two-hybrid it is demonstrated that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N-terminal of the IQSEC3 IQ-like domain. The data provide direct evidence of the interaction of IQSEC3 with GlyR-GPHN complexes, underscoring a potential role of these ArfGEFs in the function of glycinergic synapses.

中文翻译：

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使用免疫沉淀-蓝色天然凝胶电泳-质谱法分析甘氨酸受体复合物。

包含α1和β亚基的五聚体甘氨酸受体（GlyR）是脑干和脊髓中的主要抑制离子性受体。GlyR与gephyrin（GPHN）相互作用，这是一种将GlyR锚定在质膜上并使其在甘氨酸能突触后形成簇的支架蛋白。使用相互作用蛋白质组学方法，提供了ArfGEFs IQ基序和Sec7域3（IQSEC3）和IQ基序和Sec7域2（IQSEC2）作为与GlyR复合体相互作用的两种新型突触蛋白的证据。当亲和力分离的GlyR复合物通过蓝色天然凝胶电泳进行分馏并通过质谱进行表征时，GlyRα1β-GPHN表现为最丰富的复合物，分子量约为≈1MDa，GlyRα1β-GPHN-IQSEC3为次要蛋白质。约1.2 MDa的复数。第三GlyRα1β-GPHN-IQSEC2复合物以最低的量存在，其质量类似于含IQSEC3的复合物。使用酵母双杂交，证明IQSEC3通过与IQSEC3 IQ样结构域N末端的GPHN G结构域结合而与GlyR复合物相互作用。数据提供了IQSEC3与GlyR-GPHN复合物相互作用的直接证据，突显了这些ArfGEF在甘氨酸能突触功能中的潜在作用。