Ligand binding of the second PDZ domain regulates clustering of PSD-95 with the Kv1.4 potassium channel

Fumiaki Imamura, Shoji Maeda, Tomoko Doi, Yoshinori Fujiyoshi

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3640-3646
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number5
DOIs
StatePublished - Feb 1 2002

Fingerprint

Kv1.4 Potassium Channel
PDZ Domains
Cluster Analysis
Ligands
Proteins
Bearings (structural)
Guanylate Kinases
src Homology Domains
Sequence Deletion
COS Cells
Glutamate Receptors
Missense Mutation
N-Methylaspartate
N-Methyl-D-Aspartate Receptors
Ion Channels

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Imamura, Fumiaki ; Maeda, Shoji ; Doi, Tomoko ; Fujiyoshi, Yoshinori. / Ligand binding of the second PDZ domain regulates clustering of PSD-95 with the Kv1.4 potassium channel. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 5. pp. 3640-3646.
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abstract = "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.",
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Ligand binding of the second PDZ domain regulates clustering of PSD-95 with the Kv1.4 potassium channel. / Imamura, Fumiaki; Maeda, Shoji; Doi, Tomoko; Fujiyoshi, Yoshinori.

In: Journal of Biological Chemistry, Vol. 277, No. 5, 01.02.2002, p. 3640-3646.

Research output: Contribution to journalArticle

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T1 - Ligand binding of the second PDZ domain regulates clustering of PSD-95 with the Kv1.4 potassium channel

AU - Imamura, Fumiaki

AU - Maeda, Shoji

AU - Doi, Tomoko

AU - Fujiyoshi, Yoshinori

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AB - 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.

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