Department of Pharmacology, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom.
J Biol Chem. 2010 Sep 17;285(38):29623-31. doi: 10.1074/jbc.M110.121368. Epub 2010 Jul 9.
Gephyrin and collybistin are key components of GABA(A) receptor (GABA(A)R) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA(A)R subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA(A)R α2 and α3 subunit intracellular M3-M4 domain (but not α1, α4, α5, α6, β1-3, or γ1-3) with gephyrin. Curiously, GABA(A)R α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA(A)R α2 also overlap at the start of the gephyrin E domain. This suggests that although GABA(A)R α3 interacts with gephyrin, GABA(A)R α2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA(A)R α2 and collybistin or GABA(A)R α2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA(A)R α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA(A)R α2 is capable of "activating " collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA(A)R α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA(A)R and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with α2 subunit containing GABA(A)Rs, but not GlyRs or other GABA(A)R subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABA(A)Rs containing the α2 subunit.
Gephyrin 和 collybistin 是 GABA(A) 受体 (GABA(A)R) 聚集的关键组成部分。然而，解析大量 GABA(A)R 亚基与这些聚集蛋白之间的分子相互作用是一项重大挑战。我们报告了 GABA(A)R α2 和 α3 亚基细胞内 M3-M4 结构域（但不是 α1、α4、α5、α6、β1-3 或 γ1-3）与 gephyrin 的直接相互作用。奇怪的是，GABA(A)R α2 但不是 α3，使用重叠位点与 gephyrin 和 collybistin 结合。Gephyrin 上 collybistin 和 GABA(A)R α2 的相互作用结合位点也在 gephyrin E 结构域的起始处重叠。这表明，尽管 GABA(A)R α3 与 gephyrin 相互作用，但 GABA(A)R α2、collybistin 和 gephyrin 形成三聚体复合物。支持这一观点的是，在存在 gephyrin 或 collybistin 的情况下，GABA(A)R α2 与 collybistin 或 GABA(A)R α2 与 gephyrin 之间的三杂交相互作用得到增强。在共免疫沉淀实验中，collybistin 和 gephyrin 也相互竞争与 GABA(A)R α2 结合，并且在转染细胞中在细胞内和亚膜聚集体中共同定位。有趣的是，GABA(A)R α2 能够“激活”含有调节 SH3 结构域的 collybistin 同工型，从而使 gephyrin 靶向亚膜聚集体。GABA(A)R α2-collybistin 相互作用被 collybistin SH3 结构域（p.G55A）中的致病突变破坏，该突变通过破坏 GABA(A)R 和 gephyrin 聚集导致 X 连锁智力障碍和癫痫。由于视网膜免疫组织化学显示 collybistin 与含有 α2 亚基的 GABA(A)Rs 优先共定位，而不是 GlyRs 或其他 GABA(A)R 亚型，我们提出 collybistin-gephyrin 复合物在含有 α2 亚基的 GABA(A)R 聚集中具有密切作用。
