Analysis of the potential role of GluA4 carboxyl-terminus in PDZ interactions.
Analysis of the potential role of GluA4 carboxyl-terminus in PDZ interactions.
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分析GLUA4羧基末端在PDZ相互作用中的潜在作用。
DOI：
10.1371/journal.pone.0008715
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发表时间：
2010-01-14
期刊：
影响因子：
3.7
通讯作者：
Keinänen K
中科院分区：
文献类型：
--
作者：
Coleman SK;Cai C;Kalkkinen N;Korpi ER;Keinänen K
具有长尾亚基的α-氨基-3-羟基-5-甲基异恶唑-4-丙酸酯（AMPA）受体特异性传递到突触被认为是许多形式的突触强度的活性依赖性变化的关键事件。GluA 1是最具特征的长尾AMPA受体亚基，含有C-末端I类PDZ结合基序，其介导其与支架和运输蛋白的相互作用，包括突触相关蛋白97（SAP 97）。在GluA 4中，另一个参与突触可塑性的长尾亚基，PDZ基序被单个脯氨酸残基阻断。这个特征在脊椎动物中是高度保守的，而GluA 4的最接近的无脊椎动物同源物具有典型的I类PDZ结合基序。在这项工作中，我们研究了GluA 4在PDZ相互作用中的作用。删除重组GluA 4的羧基末端脯氨酸残基赋予亲和结合SAP 97在培养的细胞中所示的免疫共沉淀，而野生型GluA 4不与SAP 97。天然GluA 4和SAP 97 coimmunoprecipitated从小鼠脑独立的GluA 1亚基，支持在体内PDZ相互作用的可能性。为了获得支持或反对天然GluA 4受体的羧基末端加工暴露PDZ基序的证据，我们产生了对脯氨酸缺失的GluA 4 C-末端具有特异性的抗体试剂。免疫沉淀和质谱分析表明，天然GluA 4 AMPA受体的羧基末端是完整的，并且假定的单残基裂解不会发生任何显着的程度。我们的结论是，天然GluA 4受体是不能够典型的PDZ相互作用，它们与SAP 97的关联可能是间接的。
Specific delivery to synapses of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors with long-tailed subunits is believed to be a key event in many forms of activity-dependent changes in synaptic strength. GluA1, the best characterized long-tailed AMPA receptor subunit, contains a C-terminal class I PDZ binding motif, which mediates its interaction with scaffold and trafficking proteins, including synapse-associated protein 97 (SAP97). In GluA4, another long-tailed subunit implicated in synaptic plasticity, the PDZ motif is blocked by a single proline residue. This feature is highly conserved in vertebrates, whereas the closest invertebrate homologs of GluA4 have a canonical class I PDZ binding motif. In this work, we have examined the role of GluA4 in PDZ interactions. Deletion of the carboxy-terminal proline residue of recombinant GluA4 conferred avid binding to SAP97 in cultured cells as shown by coimmunoprecipitation, whereas wild-type GluA4 did not associate with SAP97. Native GluA4 and SAP97 coimmunoprecipitated from mouse brain independently of the GluA1 subunit, supporting the possibility of in vivo PDZ interaction. To obtain evidence for or against the exposure of the PDZ motif by carboxyterminal processing of native GluA4 receptors, we generated an antibody reagent specific for proline-deleted GluA4 C-terminus. Immunoprecipitation and mass spectrometric analyses indicated that the carboxyl-terminus of native GluA4 AMPA receptors is intact and that the postulated single-residue cleavage does not occur to any significant extent. We conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect.
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