Postsynaptic targeting of alternative postsynaptic density-95 isoforms by distinct mechanisms.
Members of the postsynaptic density-95 (PSD95)/synapse-associated protein-90 (SAP90) family of scaffolding proteins contain a common set of modular protein interaction motifs including PDZ (postsynaptic density-95/Discs large/zona occludens-1), Src homology 3, and guanylate kinase domains, which regulate signaling and plasticity at excitatory synapses. We report that N-terminal alternative splicing of PSD95 generates an isoform, PSD95beta that contains an additional "L27" motif, which is also present in SAP97. Using yeast two hybrid and coimmunoprecipitation assays, we demonstrate that this N-terminal L27 domain of PSD-95beta, binds to an L27 domain in the membrane-associated guanylate kinase calcium/calmodulin-dependent serine kinase, and to Hrs, an endosomal ATPase that regulates vesicular trafficking. By transfecting heterologous cells and hippocampal neurons, we find that interactions with the L27 domain regulate synaptic clustering of PSD95beta. Disrupting Hrs-regulated early endosomal sorting in hippocampal neurons selectively blocks synaptic clustering of PSD95beta but does not interfere with trafficking of the palmitoylated isoform, PSD95alpha. These studies identify molecular and functional heterogeneity in synaptic PSD95 complexes and reveal critical roles for L27 domain interactions and Hrs regulated vesicular trafficking in postsynaptic protein clustering.
Chetkovich
Dane M
DM
Department of Physiology, University of California, San Francisco, San Francisco, California 94143-0444, USA.
Bunn
Robert C
RC
Kuo
Sheng-Han
SH
Kawasaki
Yoshimi
Y
Kohwi
Minoree
M
Bredt
David S
DS
D048868:Adaptor Proteins, Signal Transducing; D017398:Alternative Splicing / Q000235:genetics; D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D000074288:Discs Large Homolog 1 Protein; D000076122:Disks Large Homolog 4 Protein; D056827:Endosomal Sorting Complexes Required for Transport; D051528:Guanylate Kinases; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D012377:Rodentia; D017386:Sequence Homology, Amino Acid; D013569:Synapses / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; C513314:DLG1 protein, human; C501444:DLG4 protein, human; D000074288:Discs Large Homolog 1 Protein; D000076122:Disks Large Homolog 4 Protein; C501119:Dlg1 protein, rat; C501449:Dlg4 protein, rat; D056827:Endosomal Sorting Complexes Required for Transport; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; C033652:postsynaptic density proteins; C106868:CASK kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases