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

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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.