Maximov Anton, Tang Tie Shan, Bezprozvanny Ilya
Department of Physiology, UT Southwestern Medical Center at Dallas, 75390, Dallas, TX, USA.
Mol Cell Neurosci. 2003 Feb;22(2):271-83. doi: 10.1016/s1044-7431(02)00027-1.
The type 1 inositol (1,4,5)-trisphosphate receptor (InsP(3)R1) is an intracellular calcium (Ca(2+)) release channel that plays an important role in neuronal function. In yeast two-hybrid screen of rat brain cDNA library with the InsP(3)R1 carboxy-terminal bait we isolated multiple clones of neuronal cytoskeletal protein 4.1N. We mapped the 4.1N-interaction site to a short fragment (50 amino acids) within the carboxy-terminal tail of the InsP(3)R1 and the InsP(3)R1-interaction site to the carboxy-terminal domain (CTD) of 4.1N. We established that InsP(3)R1 carboxy-terminal binds selectively to the CTDDelta alternatively spliced form of the 4.1N protein. In biochemical experiments we demonstrated that 4.1N and InsP(3)R1 specifically associate in vitro. We showed that both 4.1N and InsP(3)R1 were enriched in synaptic locations and immunoprecipitated the 4.1N-InsP(3)R1 complex from rat brain synaptosomes. In biochemical experiments we demonstrated a possibility of InsP(3)R1-4.1N-CASK-syndecan-2 quaternary complex formation. From our findings we hypothesize that InsP(3)R1-4.1N association may play a role in InsP(3)R1 localization or Ca(2+) signaling in neurons.
1型肌醇（1,4,5）-三磷酸受体（InsP(3)R1）是一种细胞内钙（Ca(2+)）释放通道，在神经元功能中起重要作用。在用InsP(3)R1羧基末端作为诱饵对大鼠脑cDNA文库进行酵母双杂交筛选时，我们分离出了多个神经元细胞骨架蛋白4.1N的克隆。我们将4.1N相互作用位点定位到InsP(3)R1羧基末端尾巴内的一个短片段（50个氨基酸），并将InsP(3)R1相互作用位点定位到4.1N的羧基末端结构域（CTD）。我们确定InsP(3)R1羧基末端选择性地与4.1N蛋白的CTDDelta可变剪接形式结合。在生化实验中，我们证明4.1N和InsP(3)R1在体外特异性结合。我们发现4.1N和InsP(3)R1在突触位置均富集，并从大鼠脑突触小体中免疫沉淀出4.1N-InsP(3)R1复合物。在生化实验中，我们证明了InsP(3)R1-4.1N-CASK- syndecan-2四元复合物形成的可能性。根据我们的研究结果，我们推测InsP(3)R1与4.1N的结合可能在神经元中InsP(3)R1的定位或Ca(2+)信号传导中发挥作用。
