Analysis of interaction between a novel PDZ protein and glutamate receptor δ subunit C-terminus.

新型 PDZ 蛋白与谷氨酸受体 δ 亚基 C 末端相互作用的分析。

基本信息

批准号：
11680610
负责人：
KAWAMOTO Susumu
金额：
$ 2.37万
依托单位：
Yokohama City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680610/
关键词：
Glutamate receptor Ion channel δ2 subunit PDZ domain PDZ protein Surface plasmon resonance Scafolding protein Protein-protein interferon

项目摘要

We identified a novel GluRδ2-interacting protein, named Delphilin (formerly δ2SIP, GenBank accession # AA869045), containing a PDZ domain. Because a PDZ domain is a binding motif generally recognizing the C-terminus of its target proteins, we assayed Delphilin interaction with various C-terminal mutants of the GluRδ2 subunit by yeast two-hybrid analysis and by surface plasmon resonance (SPR) analyses using BIAcore, which effectively monitor interactions between macromolecules in real time. In SPR experiments, a solution of a glutathione Stransferase (GST) fusion protein of Delphilin PDZ was passed over two flow cells : one immobilized with the GluRδ2 C-terminal 8-residue peptide (DPDRGTSI), and one with the GIuRδ1 corresponding peptide (DTSHGTSI). The Delphilin PDZ exhibited a specific binding to the C-terminal peptide of GluRδ2, but not of GluRδ1. From these results, it appears that the PDZ domain of Delphilin interacts directly with the C-terminus of GluRδ2 and that amino acids-6 to -4 of the C-terminus may critically contribute to the binding. In yeast two-hybrid system, Delphilin-PDZ domain, containing the adjacent area, had more affinity to the target GluRδ2 C-terminal than the original Delphilin-PDZ and the area (amino acid 227 to 352) of Delphilin seemed to have negative effect on the interaction between Delphilin-PDZ and GluRδ2 C-terminus. In SPR experiments, it was also suggested that the phosphorylation of δ2 C-terminal Ser (-1 position) or Thr (-2 position) may interefere with the interaction.

我们确定了一种新型的GlurΔ2相互作用蛋白，称为Delphilin（以前是Δ2SIP，GenBank登录＃AA869045），其中包含PDZ结构域。由于PDZ结构域是一个结合基序，通常识别其靶蛋白的C末端，因此我们通过酵母两种杂交分析和表面等离子体共振（SPR）分析使用BIAC进行了BIAC，从而分配了Delphilin与GlurΔ2亚基的各种C端突变体的相互作用，从而有效地监测了实时时代的Macromolecles之间的相互作用。在SPR实验中，Delphilin PDZ的谷胱甘肽曲ransferase（GST）融合蛋白的溶液通过了两个流动细胞：一种用GlURΔ2C末端8分子肽（DPDRGTSI）固定的，一个固定在两个流动细胞上，一种固定在GIURδ1中，用GIURδ1相应的肽（DTSHGTSSI）。 Delphilin PDZ表现出特异性结合与GlurΔ2的C末端肽，但没有GlurΔ1的结合。从这些结果中看来，Delphilin的PDZ结构域直接与GlurΔ2的C末端相互作用，并且C-末端的氨基酸-6至-4可能对结合产生了严格的贡献。在酵母双杂交系统中，包含相邻区域的Delphilin-PDZ结构域比原始的Delphilin-PDZ和Delphilin的原始Delphilin-PDZ和该区域（氨基酸227至352）具有更大的亲和力，似乎对Delphilin-PDZ和GlurUn-PDZ和GlurUeΔ2C-termerinus之间的相互作用产生了负面影响。在SPR实验中，还建议Δ2c末端Ser（-1位置）或THR（-2位置）的磷酸化可能与相互作用相互作用。