Department of Biosciences, Division of Biochemistry, Viikki Biocenter, University of Helsinki, Helsinki, Finland.
PLoS One. 2010 Jan 14;5(1):e8715. doi: 10.1371/journal.pone.0008715.
Specific delivery to synapses of alpha-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.
METHODOLOGY/PRINCIPAL FINDINGS: 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.
CONCLUSION/SIGNIFICANCE: We conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect.
人们相信，将 α-氨基-3-羟基-5-甲基异恶唑-4-丙酸（AMPA）受体的长尾亚基特异性递送至突触是许多形式的突触强度活性依赖性变化的关键事件。GluA1 是最具特征的长尾 AMPA 受体亚基，其包含 C 末端 I 类 PDZ 结合基序，该基序介导其与支架和运输蛋白（包括突触相关蛋白 97（SAP97））的相互作用。在另一个与突触可塑性相关的长尾亚基 GluA4 中，PDZ 基序被单个脯氨酸残基阻断。该特征在脊椎动物中高度保守，而 GluA4 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 GluA4 在 PDZ 相互作用中的作用。
方法/主要发现：重组 GluA4 的羧基末端脯氨酸残基缺失赋予了其在培养细胞中与 SAP97 结合的强烈亲和力，如共免疫沉淀所示，而野生型 GluA4 则不与 SAP97 结合。来自小鼠大脑的天然 GluA4 和 SAP97 独立于 GluA1 亚基共免疫沉淀，支持体内 PDZ 相互作用的可能性。为了获得关于天然 GluA4 受体的羧基末端加工是否通过 PDZ 基序暴露的证据或反对证据，我们生成了一种针对脯氨酸缺失的 GluA4 C 末端的特异性抗体试剂。免疫沉淀和质谱分析表明，天然 GluA4 AMPA 受体的羧基末端是完整的，并且推测的单个残基切割不会以任何显著程度发生。
结论/意义：我们得出结论，天然 GluA4 受体不能进行典型的 PDZ 相互作用，并且它们与 SAP97 的结合可能是间接的。
