Protein kinase Cγ associates directly with the GluR4 α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit -: Effect on receptor phosphorylation
Protein kinase Cγ associates directly with the GluR4 α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit -: Effect on receptor phosphorylation
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DOI：
10.1074/jbc.m205587200
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发表时间：
2003-02-21
影响因子：
4.8
通讯作者：
Carvalho, AL
中科院分区：
文献类型：
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作者：
Correia, SS;Duarte, CB;Carvalho, AL
离子型谷氨酸受体介导了大脑中大部分兴奋性突触传递，并被认为参与学习和记忆的形成。α-氨基-3-羟基-5-甲基-4-异恶唑丙酸酯(AMPA)型谷氨酸受体的活性可以通过其亚基的直接磷酸化来调节，从而影响受体的电生理特性，以及受体与许多调节膜运输和突触靶向的蛋白质的结合。在本研究中，我们研究了蛋白激酶C(PKC)γ亚型与GluR4 AMPA受体亚单位的关系。在大鼠小脑和培养的鸡视网膜细胞提取物中，PKCGamma与GluR4 AMPA受体亚单位共沉淀，免疫细胞化学实验表明GluR4和PKCGamma在培养的鸡视网膜神经元中共定位。下拉分析表明，天然PKCGamma与GIuR4C端膜近端结合，重组PKCGamma被GST-GluR4C端融合蛋白保留，表明该激酶直接与GluR4结合。此外，GST-GluR4 C-末端蛋白在GluR4Ser-482上被结合的激酶磷酸化，并保留在包括PKCGamma在内的融合蛋白中。与PKCγ相互作用的GluR4C-末端片段缺乏PKC磷酸化位点，对组蛋白H1被PKC磷酸化的抑制程度与PKC假底物肽19-31相同，表明PKCGamma与GluR4结合优先磷酸化GluR4，损害其他底物。此外，在GluR4转基因的人胚胎肾293T细胞中，PKCGamma的表达增加了质膜相关GluR4的数量。我们的结果表明，PKCGamma直接与GluR4结合，从而调节含有GluR4的AMPA受体的功能。
Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the brain and are thought to be involved in learning and memory formation. The activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors can be regulated by direct phosphorylation of their subunits, which affects the electrophysiological properties of the receptor, and the receptor association with numerous proteins that modulate membrane traffic and synaptic targeting of the receptor. In the present study we investigated the association of protein kinase C (PKC) gamma isoform with the GluR4 AMPA receptor subunit. PKCgamma was co-immunoprecipitated with GluR4 AMPA receptor subunit in rat cerebellum and in cultured chick retina cell extracts, and immunocytochemistry experiments showed co-localization of GluR4 and PKCgamma in cultured chick retinal neurons. Pull-down assays showed that native PKCgamma binds the GIuR4 C-terminal membrane-proximal region, and recombinant PKCgamma was retained by GST-GluR4 C-terminal fusion protein, suggesting that the kinase binds directly to GluR4. Furthermore, GST-GluR4 C-terminal protein was phosphorylated on GluR4 Ser-482 by bound kinases, retained by the fusion protein, including PKCgamma. The GluR4 C-terminal segment that interacts with PKCgamma, which lacks the PKC phosphorylation sites, inhibited histone H1 phosphorylation by PKC, to the same extent as the PKC pseudosubstrate peptide 19-31, indicating that PKCgamma bound to GluR4 preferentially phosphorylates GluR4 to the detriment of other substrates. Additionally, PKCgamma expression in GluR4 transfected human embryonic kidney 293T cells increased the amount of plasma membrane-associated GluR4. Our results suggest that PKCgamma binds directly to GluR4, thereby modulating the function of GluR4-containing AMPA receptors.