GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice
GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice
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DOI：
10.1074/jbc.m109.049700
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发表时间：
2010-07-02
影响因子：
4.8
通讯作者：
Kornau, Hans-Christian
中科院分区：
文献类型：
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作者：
Bartoi, Tudor;Rigbolt, Kristoffer T. G.;Kornau, Hans-Christian
GABA（B）受体作为神经递质γ-氨基丁酸（GABA）的异二聚体G蛋白偶联受体发挥作用。基于配体结合亚基GABA（B1）的同种型的受体亚型被认为涉及相关蛋白质的差异集。在这里，我们描述了两个小鼠线，允许一个简单的GABA（B）受体的生化分离。转基因小鼠表达GABA（B1）亚型，除了其内源性亚基库之外，还包含两步亲和纯化的序列。对转基因小鼠和野生型对照动物的纯化样品进行比较分析，发现GABA（B1）复合物的两种新组分。其中一种鉴定的蛋白质，含钾通道四聚化结构域的蛋白质12，通过GABA（B2）亚基与异源二聚体GABA（B）受体结合。在转染的海马神经元中，含钾通道四聚化结构域的蛋白12增强了GABA（B2）的轴突表面靶向。在GABA（B1）上配备标签的小鼠促进GABA（B）受体的天然结合配偶体的验证和鉴定，从而提供对突触调节的分子机制的深入了解。
GABA(B) receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter gamma-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligand-binding subunit GABA(B1), are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABA(B) receptors. The transgenic mice express GABA(B1) isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABA(B1) complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABA(B) receptors via the GABA(B2) subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABA(B2). The mice equipped with tags on GABA(B1) facilitate validation and identification of native binding partners of GABA(B) receptors, providing insight into the molecular mechanisms of synaptic modulation.