Complex Assembly And Clustering Of Synaptic Proteins

Recent studies revealed that PDZ domain-containing scaffolding proteins serve an important role in clustering of diverse molecules into large complexes3. The membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins is of central importance in regulation of protein clustering at the synapse. MAGUKs are defined by the presence of a domain homologous to the yeast guanylate kinase (GK) domain, which is catalytically inactive. The GK domain is preceded by an Src-homology-3 (SH3) domain. In addition, MAGUKs contain PDZ domains, which are named for the original proteins in which the domains were identified (PSD-95, discs large, and zona occludens 1). PDZ domains bind the carboxyl terminus of proteins or form dimers with other PDZ domain-containing proteins4. PSD-95 (postsynaptic density protein of 95 kDa) is the prototypical MAGUK, and extensive research has been conducted on this protein. PSD-95 family is encoded by four genes - PSD-95/synapse-associated protein 90 (SAP90), PSD protein of 93 kDa (PSD-93)/chapsyn-110, synapse-associated protein 102 (SAP102), and synapse-associated protein 97 (SAP97), which are characterized by three PDZ domains, in addition to the SH3 and GK domain characteristic of MAGUKs3. The presence of several protein-protein interaction domains, combined with the observation that their accumulation at the synapse precedes several other known synaptic proteins, suggested an important role for MAGUKS in the assembly of a large complex at the synapse. In addition to multiple protein-protein interaction domains, protein multimerization appears to be critical for clustering proteins into large complexes3,5. For instance, PSD-95 multimerization, mediated through the palmitoylated N-terminal cysteines, is essential for ion channel clustering. Thus, protein multimerization serves as a mechanism by which scaffolding proteins can act to increase the number of molecules assembled at specific sites, such as the PSD.

In vitro assays utilizing expression of proteins in heterologous cells provided the first direct evidence that MAGUKs have the ability to cluster ion channels and associated proteins. Early studies showed that transfection of PSD-95 into heterologous cell lines, such as COS cells, leads to a mainly diffuse cytoplasmic distribution of PSD-956-8. In contrast, when PSD-95 is co-transfected with other known binding partners, both proteins form distinct clusters, or patches on the cell membrane. This phenomenon was first demonstrated with the shaker subclass of voltage-gated potassium (K+) channels6. Heterologous expression of shaker-type subunits with PSD-95 results in the co-clustering of the two proteins. Further, subunit binding and clustering by PSD-95 is blocked by a mutation in the carboxyl terminus of the shaker subunit Kv1.4. This heterologous system for analyzing protein interaction and trafficking is highly effective, and has since been used to further study the dynamics of PSD-95 interaction and clustering. In a second set of experiments the specific PDZ domain requirements of PSD-95 for clustering of Kv1.4 were examined8. Missense and deletion mutations were introduced into the PDZ1 and/or the PDZ2 domains of full length PSD-95. Kv1.4 co-expression with the PDZ2 mutant showed a significant disruption in clustering, whereas PDZ1 mutation had little effect on channel clustering. Interestingly, a mutant containing inverted PDZ1 and PDZ2 domains also reduces clustering of the Kv1.4 channel subunits when co-expressed. Thus, the PDZ2 domain must be both intact and in the correct position in order for proper protein interaction and clustering8. Similar to clustering of K+ channel subunits, PSD-95 has also been shown to cluster several other proteins in vitro. Co-expression with PSD-95 leads to the clustering of semaphorin 4B, an integral membrane protein that participates in axon and dendrite guidance9. Studies of a similar design in COS cells show that PSD-95 induces clustering of the adhesion molecule neuroligin-1 through PDZ-mediated interactions (Figure 4.2) 7.

In addition to PSD-95, this assay has been useful in the assessment of the clustering activity of other scaffolding proteins. For example, Shank has been shown to cluster mGluR5 in heterologous cells in the presence of Homer10. Further, Shank was shown to mediate the co-clustering of Homer with a complex containing PSD-95 and GKAP. Thus, Shank may provide a link between mGluR5/Homer and PSD-95/NMDA receptor complexes at the PSD10. Further, the experiments in heterologous cell lines reveal the general role of scaffolding proteins, acting to assemble and localize protein complexes.

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