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

我们鉴定了一种新的GluRδ2相互作用蛋白，命名为Delphilin（以前称为δ2SIP，GenBank登录号AA 869045），其含有PDZ结构域。由于PDZ结构域是通常识别其靶蛋白的C-末端的结合基序，我们通过酵母双杂交分析和使用BIAcore的表面等离子体共振（SPR）分析来测定Delphilin与GluRδ2亚基的各种C-末端突变体的相互作用，其有效地监测大分子之间的相互作用，真实的时间。在SPR实验中，将Delphilin PDZ的谷胱甘肽S转移酶（GST）融合蛋白的溶液通过两个流动池：一个固定有GluRδ2 C-末端8-残基肽（DPDRGTSI），一个固定有GluR δ1相应肽（DTSHGTSI）。Delphilin PDZ与GluRδ2的C-末端肽特异性结合，但与GluRδ1的C-末端肽不特异性结合。从这些结果来看，似乎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可能对结合起关键作用。在酵母双杂交系统中，Delphilin-PDZ结构域与GluR δ 2C端的相互作用比Delphilin-PDZ结构域更强，Delphilin-PDZ结构域的227 ~ 352位氨基酸对GluR δ 2C端与Delphilin-PDZ的相互作用有负效应。在SPR实验中，还发现δ2 C端Ser（-1位）或Thr（-2位）的磷酸化可能参与了这种相互作用。

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.