Phospholipase C-related inactive protein is implicated in the constitutive internalization of GABAA receptors mediated by clathrin and AP2 adaptor complex
Phospholipase C-related inactive protein is implicated in the constitutive internalization of GABAA receptors mediated by clathrin and AP2 adaptor complex
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DOI：
10.1111/j.1471-4159.2006.04399.x
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发表时间：
2007-05-01
影响因子：
4.7
通讯作者：
Hirata, Masato
中科院分区：
文献类型：
--
作者：
Kanematsu, Takashi;Fujii, Makoto;Hirata, Masato
调节突触抑制强度的机制是通过改变细胞表面可用的GABA（A）受体的数量来实现的。网格蛋白和衔接蛋白2（AP 2）复合物介导的内吞作用在调节细胞表面GABA（A）受体数量中起重要作用。最近，我们已经阐明，磷脂酶C相关的催化失活蛋白（PRIP）分子参与磷酸化依赖性调节GABA（A）受体的内化，通过与受体β亚基和蛋白磷酸酶。在这项研究中，我们研究了PRIP分子在网格蛋白介导的组成型GABA（A）受体内吞作用中的意义，独立于磷酸化调节。我们使用瞬时表达GABA（A）受体α/β/γ亚基和PRIP的人胚肾293（HEK 293）细胞进行组成型受体内化测定。PRIP与GABA（A）受体一起被内化，并且该过程被阻断PRIP与β亚基结合的PRIP结合肽抑制。网格蛋白重链、AP 2和PRIP-1的μ 2和β 2亚基与脑提取物中的GABA（A）受体复合，如通过使用抗PRIP-1和抗β 2/3 GABA（A）受体抗体的免疫共沉淀测定或通过使用GABA（A）受体的β亚基的下拉测定所分析。这些结果表明PRIP主要参与GABA（A）受体的组成性内化，其需要网格蛋白和AP 2蛋白复合物。
A mechanism for regulating the strength of synaptic inhibition is enabled by altering the number of GABA(A) receptors available at the cell surface. Clathrin and adaptor protein 2 (AP2) complex-mediated endocytosis is known to play a fundamental role in regulating cell surface GABA(A) receptor numbers. Very recently, we have elucidated that phospholipase C-related catalytically inactive protein (PRIP) molecules are involved in the phosphorylation-dependent regulation of the internalization of GABA(A) receptors through association with receptor beta subunits and protein phosphatases. In this study, we examined the implications of PRIP molecules in clathrin-mediated constitutive GABA(A) receptor endocytosis, independent of phospho-regulation. We performed a constitutive receptor internalization assay using human embryonic kidney 293 (HEK293) cells transiently expressed with GABA(A) receptor alpha/beta/gamma subunits and PRIP. PRIP was internalized together with GABA(A) receptors, and the process was inhibited by PRIP-binding peptide which blocks PRIP binding to beta subunits. The clathrin heavy chain, mu 2 and beta 2 subunits of AP2 and PRIP-1, were complexed with GABA(A) receptor in brain extract as analyzed by co-immunoprecipitation assay using anti-PRIP-1 and anti-beta 2/3 GABA(A) receptor antibody or by pull-down assay using beta subunits of GABA(A) receptor. These results indicate that PRIP is primarily implicated in the constitutive internalization of GABA(A) receptor that requires clathrin and AP2 protein complex.