Essential contribution of the ligand-binding beta B/beta C loop of PDZ1 and PDZ2 in the regulation of postsynaptic clustering, scaffolding, and localization of postsynaptic density-95.
Postsynaptic density-95 (PSD-95), a PSD-95/Discs large/zona occludens-1 (PDZ) domain-containing scaffold protein, clusters many signaling molecules near NMDA-type glutamate receptors in the postsynaptic densities. Although the synaptic localization of PSD-95 requires palmitoylation of two cysteines at the N terminus and the presence of at least one PDZ domain, how the clustering of PSD-95 is initiated and regulated remains essentially unknown. To address this issue, we examined PSD-95 clustering in primary cultured hippocampal neurons expressing full-length PSD-95 mutant proteins lacking the ligand-binding ability of PDZ1, PDZ2, and/or PDZ3. The formation of either excitatory or inhibitory synapses was unaffected. Combinations of individual mutations, however, significantly reduced the PSD-95 clustering index, in an approximately additive manner. The sensitivity to 2-bromo-palmitate and latrunculin A, reagents known to affect PSD-95 turnover, was also augmented. Furthermore, the synaptic recruitment of a PSD-95 ligand, synaptic GTPase-activating protein (synGAP), was significantly impaired, whereas the clustering of other scaffolding proteins, such as Homer 1c, Shank/Synamon, and PSD-93/Chapsin-110 was spared. Intriguingly, overexpression of the PSD-95 PDZ1/2/3 mutants caused the PSD-95 clusters to localize away from the dendritic shaft, resulting in the formation of elongated spines, in an inverse correlation with the overall PDZ-ligand affinity. Expression of a mutant synGAP lacking the PDZ-binding motif replicated both the clustering and spine morphology phenotypes. In conclusion, the ligand-binding affinity of the PDZ domains of PSD-95, contributed in part via its interaction with the C-terminal end of synGAP, plays a critical role in titrating the synaptic clustering of PSD-95 and controlling its tight association with the PSD scaffold, thereby affecting synapse maturation.
Nonaka
Mio
M
Department of Biophysics, Kyoto University Graduate School of Science, Kyoto 606-8502, Japan.
Doi
Tomoko
T
Fujiyoshi
Yoshinori
Y
Takemoto-Kimura
Sayaka
S
Bito
Haruhiko
H
D000818:Animals; D002452:Cell Count; D002478:Cells, Cultured; D000076122:Disks Large Homolog 4 Protein; D051528:Guanylate Kinases; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D013569:Synapses / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D000076122:Disks Large Homolog 4 Protein; C501447:Dlg4 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; D051528:Guanylate Kinases