PDZ domains are protein-interaction domains that are often found in multi-domain scaffolding proteins. PDZ-containing scaffolds assemble specific proteins into large molecular complexes at defined locations in the cell. In the postsynaptic density of neuronal excitatory synapses, PDZ proteins such as PSD-95 organize glutamate receptors and their associated signalling proteins and determine the size and strength of synapses. PDZ scaffolds also function in the dynamic trafficking of synaptic proteins by assembling cargo complexes for transport by molecular motors. As key organizers that control synaptic protein composition and structure, PDZ scaffolds are themselves highly regulated by synthesis and degradation, subcellular distribution and post-translational modification.

Single-particle electron microscopy (EM) combined with biochemical measurements revealed the molecular shape of SAP97 and a monomer-dimer transition that depended on the N-terminal L27 domain. Overexpression of SAP97 drove GluR1 to synapses, potentiated AMPA receptor (AMPAR) excitatory postsynaptic currents (EPSCs), and occluded LTP. Synaptic potentiation and GluR1 delivery were dissociable by L27 domain mutants that inhibit multimerization of SAP97. Loss of potentiation was correlated with faster turnover of monomeric SAP97 mutants in dendritic spines. We propose that L27-mediated interactions of SAP97 with itself or other proteins regulate the synaptic delivery of AMPARs. RNAi knockdown of endogenous PSD-95 depleted surface GluR1 and impaired AMPA EPSCs. In contrast, RNAi knockdown of endogenous SAP97 reduced surface expression of both GluR1 and GluR2 and inhibited both AMPA and NMDA EPSCs. Thus SAP97 has a broader role than its close relative, PSD-95, in the maintenance of synaptic function.

The synthesis of a difluorofluorescein monocarboxaldehyde platform and its use for preparing ZP8, a new member of the Zinpyr family of neuronal Zn(2+) sensors, are described. By combining an aniline photoinduced electron transfer (PET) switch and an electron-withdrawing fluorescein scaffold, ZP8 displays reduced background fluorescence and improved dynamic range compared to previous ZP probes. The bright sensor undergoes an 11-fold increase in fluorescence intensity upon Zn(2+) complexation (Phi = 0.03-0.35) with high selectivity over cellular concentrations of Ca(2+) and Mg(2+). In addition, sensors in the ZP family have been utilized for optical imaging in biological samples using two-photon microscopy (TPM). The cell-permeable ZP3 probe is capable of identifying natural pools of labile Zn(2+) within the mossy fiber synapses of live hippocampal slices using TPM, establishing the application of this technique for monitoring endogenous Zn(2+) stores.

The proper intracellular distribution of mitochondria is assumed to be critical for normal physiology of neuronal cells, but direct evidence for this idea is lacking. Extension or movement of mitochondria into dendritic protrusions correlates with the development and morphological plasticity of spines. Molecular manipulations of dynamin-like GTPases Drp1 and OPA1 that reduce dendritic mitochondria content lead to loss of synapses and dendritic spines, whereas increasing dendritic mitochondrial content or mitochondrial activity enhances the number and plasticity of spines and synapses. Thus, the dendritic distribution of mitochondria is essential and limiting for the support of synapses. Reciprocally, synaptic activity modulates the motility and fusion/fission balance of mitochondria and controls mitochondrial distribution in dendrites.

PDZ domains bind to short segments within target proteins in a sequence-specific fashion. Glutamate receptor-interacting protein (GRIP)/ABP family proteins contain six to seven PDZ domains and interact via the sixth PDZ domain (class II) with the C termini of various proteins including liprin-alpha. In addition the PDZ456 domain mediates the formation of homo- and heteromultimers of GRIP proteins. To better understand the structural basis of peptide recognition by a class II PDZ domain and PDZ-mediated multimerization, we determined the crystal structures of the GRIP1 PDZ6 domain alone and in complex with a synthetic C-terminal octapeptide of human liprin-alpha at resolutions of 1.5 and 1.8 A, respectively. Remarkably, unlike other class II PDZ domains, Ile-736 at alphaB5 rather than conserved Leu-732 at alphaB1 makes a direct hydrophobic contact with the side chain of the Tyr at the -2 position of the ligand. Moreover, the peptide-bound structure of PDZ6 shows a slight reorientation of helix alphaB, indicating that the second hydrophobic pocket undergoes a conformational adaptation to accommodate the bulkiness of the Tyr side chain, and forms an antiparallel dimer through an interface located at a site distal to the peptide-binding groove. This configuration may enable formation of GRIP multimers and efficient clustering of GRIP-binding proteins.

PDZ domain proteins play critical roles in binding, clustering and subcellular targeting of membrane receptors and ion channels. PDZ domains in multi-PDZ proteins often are arranged in groups with highly conserved spacing and intervening sequences; however, the functional significance of such tandem arrangements of PDZs is unclear. We have solved the three-dimensional structure of the first two PDZ domains of postsynaptic density protein-95 (PSD-95 PDZ1 and PDZ2), which are closely linked to each other in the PSD-95 family of scaffold proteins. The two PDZs have limited freedom of rotation and their C-terminal peptide-binding grooves are aligned with each other with an orientation preference for binding to pairs of C termini extending in the same direction. Increasing the spacing between PDZ1 and PDZ2 resulted in decreased binding between PDZ12 and its dimeric targets. The same mutation impaired the functional ability of PSD-95 to cluster Kv1.4 potassium channels in heterologous cells. The data presented provide a molecular basis for preferential binding of PSD-95 to multimeric membrane proteins with appropriate C-terminal sequences.

The membrane-associated guanylate kinase PSD-95 scaffolds N-methyl-d-aspartate receptors to cytoplasmic signaling molecules, and associates with other glutamate receptors at central synapses. However, regulation of PSD-95 in vivo is poorly understood. We provide evidence of an activity-dependent redistribution of PSD-95 to dendrites in central visual neurons that is tied to eye opening. Six hours after eye opening, increased dendritic PSD-95 coimmunoprecipitates with the same proportions of stargazin, increased proportions of the N-methyl-d-aspartate receptor subunit NR2A, and decreased proportions of NR2B. Sustained high levels of PSD-95 in dendrites are dependent on continued pattern vision in juvenile but not mature animals, suggesting that the stabilization of PSD-95 at synapses may be involved in the control of developmental plasticity.

Liprin-alpha/SYD-2 is a multimodular scaffolding protein important for presynaptic differentiation and postsynaptic targeting of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid glutamate receptors. However, the molecular mechanisms underlying these functions remain largely unknown. Here we report that liprin-alpha interacts with the neuron-specific kinesin motor KIF1A. KIF1A colocalizes with liprin-alpha in various subcellular regions of neurons. KIF1A coaccumulates with liprin-alpha in ligated sciatic nerves. KIF1A cofractionates and coimmunopreciptates with liprin-alpha and various liprin-alpha-associated membrane, signaling, and scaffolding proteins including alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors, GRIP/ABP, RIM, GIT1, and beta PIX. These results suggest that liprin-alpha functions as a KIF1A receptor, linking KIF1A to various liprin-alpha-associated proteins for their transport in neurons.

The Shank/ProSAP family of multidomain proteins is known to play an important role in organizing synaptic multiprotein complexes. Here we report a novel interaction between Shank and beta PIX, a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases. This interaction is mediated by the PDZ domain of Shank and the C-terminal leucine zipper domain and the PDZ domain-binding motif at the extreme C terminus of beta PIX. Shank colocalizes with beta PIX at excitatory synaptic sites in cultured neurons. In brain, Shank forms a complex with beta PIX and beta PIX-associated signaling molecules including p21-associated kinase (PAK), an effector kinase of Rac1/Cdc42. Importantly, overexpression of Shank in cultured neurons promotes synaptic accumulation of beta PIX and PAK. Considering the involvement of Rac1 and PAK in spine dynamics, these results suggest that Shank recruits beta PIX and PAK to spines for the regulation of postsynaptic structure.

Liprin-alpha is a multidomain protein that interacts with the LAR family of receptor protein tyrosine phosphatases and the GRIP/ABP family of AMPA receptor-interacting proteins. Previous studies have indicated that liprin-alpha regulates the development of presynaptic active zones and that the association of liprin-alpha with GRIP is required for postsynaptic targeting of AMPA receptors. However, the underlying molecular mechanisms are not well understood. Here we report that liprin-alpha directly interacts with GIT1, a multidomain protein with GTPase-activating protein activity for the ADP-ribosylation factor family of small GTPases known to regulate protein trafficking and the actin cytoskeleton. Electron microscopic analysis indicates that GIT1 distributes to the region of postsynaptic density (PSD) as well as presynaptic active zones. GIT1 is enriched in PSD fractions and forms a complex with liprin-alpha, GRIP, and AMPA receptors in brain. Expression of dominant-negative constructs interfering with the GIT1-liprin-alpha interaction leads to a selective and marked reduction in the dendritic and surface clustering of AMPA receptors in cultured neurons. These results suggest that the GIT1-liprin-alpha interaction is required for AMPA receptor targeting and that GIT1 may play an important role in the organization of presynaptic and postsynaptic multiprotein complexes.