Gephyrin is a multifunctional scaffolding protein anchoring glycine- and subtypes of GABA type A- receptors at inhibitory postsynaptic membrane specializations by binding to the microtubule (MT) and/or the actin cytoskeleton. However, the conditions under which gephyrin can bind to MTs and its regulation are currently unknown. Here, we demonstrate that during the purification of MTs from rat brain by sedimentation of polymerized tubulin using high-speed centrifugation a fraction of gephyrin was bound to MTs, whereas gephyrin phosphorylated at the CDK5-dependent site Ser270 was detached from MTs and remained in the soluble protein fraction. Moreover, after collybistin fostered phosphorylation at Ser270 the binding of a recombinant gephyrin to MTs was strongly reduced in co-sedimentation assays. Correspondingly, upon substitution of wild-type gephyrin with recombinant gephyrin carrying alanine mutations at putative CDK5 phosphorylation sites the binding of gephyrin to MTs was increased. Furthermore, the analysis of cultured HEK293T and U2OS cells by immunofluorescence-microscopy disclosed a dispersed and punctuated endogenous gephyrin immunoreactivity co-localizing with MTs which was evidently not phosphorylated at Ser270. Thus, our study provides additional evidence for the binding of gephyrin to MTs in brain tissue and in in vitro cell systems. More importantly, our findings indicate that gephyrin-MT binding is restricted to a specific gephyrin fraction and depicts phosphorylation of gephyrin as a regulatory mechanism of this process by showing that soluble gephyrin detached from MTs can be detected specifically with the mAb7a antibody, which recognizes the Ser270 phosphorylated- version of gephyrin.

Gephyrin 是一种多功能支架蛋白，通过与微管 (MT) 和/或肌动蛋白细胞骨架结合，在抑制性突触后膜特化处锚定甘氨酸和 GABA A 型受体亚型。然而，gephyrin 可以与 MT 结合的条件及其调节目前尚不清楚。在这里，我们证明了在使用高速离心通过聚合微管蛋白沉淀从大鼠脑中纯化 MTs 的过程中，一小部分 gephyrin 与 MTs 结合，而在 CDK5 依赖性位点 Ser270 磷酸化的 gephyrin 与 MTs 分离并保留在可溶性蛋白质部分。此外，在 collybistin 促进 Ser270 磷酸化后，重组 gephyrin 与 MT 的结合在共沉淀试验中显着降低。相应地，在用在推定的 CDK5 磷酸化位点携带丙氨酸突变的重组 gephyrin 替换野生型 gephyrin 后，gephyrin 与 MT 的结合增加。此外，通过免疫荧光显微镜对培养的 HEK293T 和 U2OS 细胞的分析揭示了与 MT 共定位的分散和间断的内源性 gephyrin 免疫反应性，其显然未在 Ser270 处磷酸化。因此，我们的研究为脑组织和体外细胞系统中 gephyrin 与 MT 的结合提供了额外的证据。更重要的是，我们的研究结果表明，gephyrin-MT 结合仅限于特定的 gephyrin 部分，并且通过显示从 MT 分离的可溶性 gephyrin 可以用 mAb7a 抗体特异性检测，将 gephyrin 的磷酸化描述为该过程的调节机制，