The molecular mechanisms underlying the protein assembly at synaptic junctions are thought to be important for neural functions. PSD-95, one of the major postsynaptic density proteins, is composed of three PDZ domains (PDZ1, PDZ2, and PDZ3), an SH3 domain, and a GK (guanylate kinase) domain. It binds to the N-methyl-D-aspartate glutamate receptor NR2 subunit or to the Shaker-type K+ channel, Ky1.4, via the PDZ1 or PDZ2 domain, whereas PDZ3 binds to distinct partners. The intramolecular interaction of these multiple domains has been implicated in efficient protein clustering. We introduced missense and deletion mutations into PDZ1 (PDZ1mΔ) and/or PDZ2 (PDZ2mΔ) of the full-length PSD-95 to disrupt the association of each domain with the target proteins, while preserving the overall structure. The ion channel clustering activities of the PSD-95 mutants were analyzed in COS-1 cells coexpressing each mutant and Ky1.4. The mutant bearing the dysfunctional PDZ2 (PSD-95:1-2mΔ) showed significantly reduced clustering efficiency, whereas the mutant with the dysfunctional PDZ1 (PSD-95:1mΔ-2) exhibited activity comparable with the wild-type activity. Furthermore, we also examined the requirements for the position of PDZ2 in full-length PSD-95 by constructing a series of PDZ1-PDZ2 inversion mutants. Surprisingly, the clustering activity of PSD-95:2-1mΔ was severely defective. Taken together, these findings show that PDZ2, which is endowed with the highest affinity for Ky1.4, is required for efficient ligand binding. In addition, the ligand binding at the position of the second PDZ domain in full-length PSD-95 is prerequisite for efficient and typical cluster formation. This study suggests that the correct placement of the multiple domains in the full-length PSD-95 protein is necessary for the optimal protein activity.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology