Postsynaptic Targeting of Alternative Postsynaptic Density-95 Isoforms by Distinct Mechanisms

Abstract

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.

The tetrameric L27 domain complex as an organization platform for supramolecular assemblies

“…3c). This L27 domain-mediated polymerization model is consistent with experimental observations showing that L27 domain-containing scaffold proteins, such as SAP97 and mLin-2, often form large clusters 2,3,11,13,18,19 . During this L27 domainmediated scaffold protein assembly, other protein-binding domains (such as the PDZ and SH3 domains) would be free to recruit their specific target proteins (receptors, ion channels and their downstream partners).…”

“…Consistent with earlier reports, mixing experiments using 15 N-labeled recombinant proteins showed that the L27S of SAP97 and L27N of mLin-2 formed a stable heteromeric complex and that the L27 domain of mLin-7 and L27C of mLin-2 formed another heteromeric complex 3,[13][14][15] (data not shown). We chose the L27S-L27N pair for further structural analysis owing to the better spectral quality of this complex ( Supplementary Fig.…”

“…Approximate interproton distance restraints were derived from NOESY spectra (a 1 H 2D homonuclear NOESY, a 15 N separated-NOESY and a 13 Cseparated NOESY). NOEs between two SAP97-mLin-2 L27 domain heterodimers were identified using a 13 C-edited (F1), 13 C, 15 N-filtered (F3) 3D NOESY spectrum on a 13 C, 15 N-labeled and nonlabeled single-chain fusion protein mixture (1:1) (ref. 24).…”

“…The C-terminal L27 domain of mLin-2 specifically binds to the L27 domain of 15). The discovery of L27 domains as specific protein-protein interaction modules capable of forming heteromeric complexes suggests that L27 domains can integrate multiple scaffold proteins into supramolecular assemblies 1,3,13,15,16 . However, the molecular basis of the L27 domain-mediated protein assembly formation remains unclear.…”

“…The use of this protein provided another advantage in the complex structural determination by NMR, as the L27S-L27N heterotetramer was 'decomposed' into a 'homodimer' consisting of two units of L27S-L27N. Intersubunit NOEs between two units of L27S-L27N could be reliably identified using a 13 C-edited (F1), 13 C, 15 N-filtered (F3) 3D NOESY spectrum on a 13 C, 15 N-labeled and nonlabeled protein mixture.…”