At synaptic junctions, specialized subcellular structures occur in both pre- and postsynaptic cells. Most presynaptic termini contain electron-dense membrane structures, often referred to as active zones, which function in vesicle docking and release. The components of those active zones and how they are formed are largely unknown. This study reports that a mutation in the C. elegans syd-2 (for synapse-defective) gene causes a diffused localization of several presynaptic proteins and of a synaptic-vesicle membrane associated green fluorescent protein (GFP) marker. Ultrastructural analysis revealed that the active zones of syd-2 mutants are significantly lengthened, whereas the total number of vesicles per synapse and the number of vesicles at the prominent active zones are comparable to those in wild-type animals. Synaptic transmission is partially impaired in syd-2 mutants. syd-2 encodes a member of the liprin (for LAR-interacting protein) family of proteins which interact with LAR-type (for leukocyte common antigen related) receptor proteins with tyrosine phosphatase activity (RPTPs). SYD-2 protein is localized at presynaptic termini independently of the presence of vesicles, and functions cell autonomously. It is proposed that SYD-2 regulates the differentiation of presynaptic termini in particular the formation of the active zone, by acting as an intracellular anchor for RPTP signalling at synaptic junctions (Zhen, 1999).
Active zones are presynaptic regions where synaptic vesicles fuse with plasma membrane to release neurotransmitters. Active zones are highly organized structurally and are functionally conserved among different species. Synapse defective-2 (SYD-2) family proteins regulate active zone morphology in C. elegans and Drosophila. This study demonstrates by immunoelectron microscopy that at C. elegans synapses, SYD-2 localizes strictly at active zones and can be used as an active zone marker when fused to green fluorescent protein (GFP). By driving expression of SYD-2::GFP fusion protein in GABAergic neurons, it is possible to visualize discrete fluorescent puncta corresponding to active zones in living C. elegans. During development, the number of GABAergic synapses made by specific motoneurons increases only slightly from larvae to adult stages. In contrast, the number of SYD-2::GFP puncta doubles, suggesting that individual synapses accommodate the increasing size of their synaptic targets mainly by incorporating more active zone materials. Furthermore, this marker was used to perform a genetic screen to identify genes involved in the development of active zones. 16 mutants were recovered with altered SYD-2::GFP expression, including alleles of five genes that have been implicated previously in synapse formation or nervous-system development. Mapping of 11 additional mutants suggests that they may represent novel genes involved in active zone formation (Yeh, 2005).
Leukocyte-common antigen related (LAR)-like phosphatase receptors are conserved cell adhesion molecules that function in multiple developmental processes. The Caenorhabditis elegans ptp-3 gene encodes two LAR family isoforms that differ in the extracellular domain. The long isoform, PTP-3A, localizes specifically at synapses and the short isoform, PTP-3B, is extrasynaptic. Mutations in ptp-3 cause defects in axon guidance that can be rescued by PTP-3B but not by PTP-3A. Mutations that specifically affect ptp-3A do not affect axon guidance but instead cause alterations in synapse morphology. Genetic double-mutant analysis is consistent with ptp-3A acting with the extracellular matrix component nidogen, nid-1, and the intracellular adaptor α-liprin, syd-2. nid-1 and syd-2 are required for the recruitment and stability of PTP-3A at synapses, and mutations in ptp-3 or nid-1 result in aberrant localization of SYD-2. Overexpression of PTP-3A is able to bypass the requirement for nid-1 for the localization of SYD-2 and RIM. It is proposed that PTP-3A acts as a molecular link between the extracellular matrix and α-liprin during synaptogenesis (Ackley, 2005).
The presynaptic regions of axons accumulate synaptic vesicles, active zone proteins and periactive zone proteins. However, the rules for orderly recruitment of presynaptic components are not well understood. This study systematically examined molecular mechanisms of presynaptic development in egg-laying synapses of C. elegans, demonstrating that two scaffolding molecules, SYD-1 and SYD-2, have key roles in presynaptic assembly. SYD-2 (liprin-alpha) regulate the size and the shape of active zones. In syd-1 and syd-2 mutants, synaptic vesicles and numerous other presynaptic proteins fail to accumulate at presynaptic sites. SYD-1 and SYD-2 function cell-autonomously at presynaptic terminals, downstream of synaptic specificity molecule SYG-1. SYD-1 is likely to act upstream of SYD-2 to positively regulate its synaptic assembly activity. These data imply a hierarchical organization of presynaptic assembly, in which transmembrane specificity molecules initiate synaptogenesis by recruiting a few key scaffolding proteins, which in turn assemble other presynaptic components (Patel, 2006).
A central event in synapse development is formation of the presynaptic active zone in response to positional cues. Three active zone proteins, RIM, ELKS (also known as ERC or CAST) and Liprin-alpha, bind each other and are implicated in linking active zone formation to synaptic vesicle release. Loss of function in C. elegans syd-2 Liprin-alpha alters the size of presynaptic specializations and disrupts synaptic vesicle accumulation. A missense mutation in the coiled-coil domain of SYD-2 causes a gain of function. In HSN synapses, the syd-2gf mutation promotes synapse formation in the absence of syd-1, which is essential for HSN synapse formation. syd-2gf also partially suppresses the synaptogenesis defects in syg-1 and syg-2 mutants. The activity of syd-2gf requires elks-1, an ELKS homolog; but not unc-10, a RIM homolog. The mutant SYD-2 shows increased association with ELKS. These results establish a functional dependency for assembly of the presynaptic active zone in which SYD-2 plays a key role (Dai, 2006).
LAR family transmembrane protein-tyrosine phosphatases function in axon guidance and mammary gland development. In cultured cells, LAR binds to the intracellular, coiled coil LAR-interacting protein at discrete ends of focal adhesions, implicating these proteins in the regulation of cell-matrix interactions. Seven LAR-interacting protein-like genes are described in humans and Caenorhabditis elegans that form the liprin gene family. Based on sequence similarities and binding characteristics, liprins may be subdivided into either α- or beta-type. The C-terminal, non-coiled coil regions of α-liprins bind to the membrane-distal phosphatase domains of LAR family members, as well as to the C-terminal, non-coiled coil region of beta-liprins. Both α- and beta-liprins homodimerize via their N-terminal, coiled coil regions. Liprins are thus multivalent proteins that potentially form complex structures. Some liprins have broad mRNA tissue distributions, whereas others are predominately expressed in the brain. Co-expression studies indicate that liprin-α2 alters LAR cellular localization and induces LAR clustering. It is proposed that liprins function to localize LAR family tyrosine phosphatases at specific sites on the plasma membrane, possibly regulating their interaction with the extracellular environment and their association with substrates (Serra-Pages, 1998).
Interaction with the multi-PDZ protein GRIP is required for the synaptic targeting of AMPA receptors. GRIP binds to the liprin-α/SYD2 family of proteins that interacts with LAR receptor protein tyrosine phosphatases (LAR-RPTPs) that are implicated in presynaptic development. In neurons, liprin-α and LAR-RPTP are enriched at synapses and coimmunoprecipitate with GRIP and AMPA receptors. Dominant-negative constructs that interfere with the GRIP-liprin interaction disrupt the surface expression and dendritic clustering of AMPA receptors in cultured neurons. Thus, by mediating the targeting of liprin/GRIP-associated proteins, liprin-α is important for postsynaptic as well as presynaptic maturation (Wyszynski, 2002).
Metastasis-associated protein S100A4 (Mts1) induces invasiveness of primary tumors and promotes metastasis. S100A4 belongs to the family of small calcium-binding S100 proteins that are involved in different cellular processes as transducers of calcium signal. S100A4 modulates properties of tumor cells via interaction with its intracellular targets, heavy chain of non-muscle myosin and p53. A new molecular target of the S100A4 protein has been identified, liprin beta1. Liprin beta1 belongs to the family of leukocyte common antigen-related (LAR) transmembrane tyrosine phosphatase-interacting proteins that may regulate LAR protein properties via interaction with another member of the family, liprin alpha1. This study shows by the immunoprecipitation analysis that S100A4 interacts specifically with liprin beta1 in vivo. Immunofluorescence staining demonstrated the co-localization of S100A4 and liprin beta1 in the cytoplasm and particularly at the protrusion sites of the plasma membrane. The S100A4 binding site maps at the C terminus of the liprin beta1 molecule between amino acid residues 938 and 1005. The S100A4-binding region contains two putative phosphorylation sites by protein kinase C and protein kinase CK2. S100A4-liprin beta1 interaction results in the inhibition of liprin beta1 phosphorylation by both kinases in vitro (Kriajevska, 2002).
Liprin-α 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-α regulates the development of presynaptic active zones and that the association of liprin-α with GRIP is required for postsynaptic targeting of AMPA receptors. However, the underlying molecular mechanisms are not well understood. Liprin-α 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-α, GRIP, and AMPA receptors in brain. Expression of dominant-negative constructs interfering with the GIT1-liprin-α 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-α interaction is required for AMPA receptor targeting and that GIT1 may play an important role in the organization of presynaptic and postsynaptic multiprotein complexes (Ko, 2003a).
Liprin-alpha/SYD-2 is a family of multidomain proteins with four known isoforms. One of the reported functions of liprin-alpha is to regulate the development of presynaptic active zones, but the underlying mechanism is poorly understood. This study reports that liprin-alpha directly interacts with the ERC (ELKS-Rab6-interacting protein-CAST) family of proteins, members of which are known to bind RIMs, the active zone proteins that regulate neurotransmitter release. In vitro results indicate that ERC2/CAST, an active zone-specific isoform, interacts with all of the known isoforms of liprin-alpha and that liprin-alpha1 associates with both ERC2 and ERC1b, a splice variant of ERC1 that distributes to both cytosolic and active zone regions. ERC2 colocalizes with liprin-alpha1 in cultured neurons and forms a complex with liprin-alpha1 in brain. Liprin-alpha1, when expressed alone in cultured neurons, shows a partial synaptic localization. When coexpressed with ERC2, however, liprin-alpha1 is redistributed to synaptic sites. Moreover, roughly the first half of ERC2, which contains the liprin-alpha-binding region, is sufficient for the synaptic localization of liprin-alpha1 while the second half is not. These results suggest that the interaction between ERC2 and liprin-alpha may be involved in the presynaptic localization of liprin-alpha and the molecular organization of presynaptic active zones (Ko, 2003b).
Liprin-alpha/SYD-2 is a multimodular scaffolding protein important for presynaptic differentiation and postsynaptic targeting of AMPA glutamate receptors. However, the molecular mechanisms underlying these functions remain largely unknown. This study reports 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 AMPA 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 (Shin, 2003).
The LAR transmembrane tyrosine phosphatase associates with liprin-α proteins and colocalizes with liprin-α1 at focal adhesions. LAR has been implicated in axon guidance, and liprins are involved in synapse formation and synapse protein trafficking. Several liprin mutants have weaker binding to LAR as assessed by yeast interaction trap assays, and the extent of in vitro and in vivo phosphorylation of these mutants was reduced relative to that of wild-type liprin-α1. Treatment of liprin-α1 with calf intestinal phosphatase weakens its interaction with the recombinant GST-LAR protein. A liprin LH region mutant that inhibits liprin phosphorylation does not bind to LAR as assessed by coprecipitation studies. Endogenous LAR binds phosphorylated liprin-α1 from MDA-486 cells labeled in vivo with [32P]orthophosphate. In further characterizing the phosphorylation of liprin, immunoprecipitates of liprin-α1 expressed in COS-7 cells were found to incorporate phosphate after washes of up to 4 M NaCl. Additionally, purified liprin-α1 derived from Sf-9 insect cells retains the ability to incorporate phosphate in in vitro phosphorylation assays, and a liprin-α1 truncation mutant incorporates phosphate after denaturation and/or renaturation in SDS gels. Finally, binding assays show that liprin binds to ATP-agarose and that the interaction is challenged by free ATP, but not by free GTP. Moreover, liprin LH region mutations that inhibit liprin phosphorylation stabilize the association of liprin with ATP-agarose. Taken together, these results suggest that liprin autophosphorylation regulates its association with LAR (Serra-Pages, 2005).
Leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (LAR-RPTP) bind to liprin-alpha (SYD2) and are implicated in axon guidance. LAR-RPTP is concentrated in mature synapses in cultured rat hippocampal neurons, and is important for the development and maintenance of excitatory synapses in hippocampal neurons. RNA interference (RNAi) knockdown of LAR or dominant-negative disruption of LAR function results in loss of excitatory synapses and dendritic spines, reduction of surface AMPA receptors, impairment of dendritic targeting of the cadherin-beta-catenin complex, and reduction in the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Cadherin, beta-catenin and GluR2/3 are tyrosine phosphoproteins that coimmunoprecipitate with liprin-alpha and GRIP from rat brain extracts. It is proposed that the cadherin-beta-catenin complex is cotransported with AMPA receptors to synapses and dendritic spines by a mechanism that involves binding of liprin-alpha to LAR-RPTP and tyrosine dephosphorylation by LAR-RPTP (Dunah, 2005).
Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. The MALS protein complex was purified from brain and it was found liprin-alpha as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS-CASK-liprin-alpha complex recruits components of the synaptic release machinery to adhesive proteins of the active zone (Olsen, 2005).
The ternary scaffolding protein complex of MALS/Veli (mammalian LIN-7/vertebrate homologue of LIN-7), CASK (peripheral plasma membrane protein), and Mint-1 (munc-18 interacting protein 1), consist of vertebrate homologues of a complex first identified in Caenorhabditis elegans that mediates vulval development. In mammalian brain, the MALS–CASK–Mint-1 complex occurs on both sides of synaptic junctions and is thought to serve distinct roles in these two locations. Presynaptically, this complex links to neurexin, an adhesion molecule that binds across the synapse to postsynaptic neuroligin. Furthermore, Mint-1 associates with Munc18-1, an essential component of the synaptic vesicle fusion machinery. Postsynaptically, MALS binds to the N-methyl-D-aspartate (NMDA)–type of glutamate receptors and is reported to transport NMDA receptor vesicles along microtubules (Olsen, 2005 and references therein).
Genetic studies have failed to establish the essential roles of the MALS–CASK–Mint-1 complex in brain. Three MALS genes exist in mammals, and targeted disruption of MALS-1 and MALS-2 leads to compensatory up-regulation of MALS-3 in the CNS. Mint-1 mutant mice show no defects in excitatory synaptic transmission and only a subtle defect in inhibitory synaptic transmission . Also, no synaptic analysis has been reported for CASK knockouts that die at birth due to midline defects (Olsen, 2005 and references therein).
Several molecules that mediate synapse development have been identified through invertebrate genetic studies. For example, mutation of C. elegans syd-2 disperses presynaptic active zones (Zhen, 1999). A similar structural defect occurs in flies lacking the Drosophila melanogaster syd orthologue liprin-alpha, which exhibits a concomitant decrease in synaptic transmission (Kaufmann, 2002). Liprin-α binds to a receptor protein tyrosine phosphatase, Dlar (Serra-Pages, 1998), suggesting a model whereby liprin-α and Dlar cooperate to organize presynaptic active zones. How liprin-α links to the synaptic vesicle machinery remains uncertain (Olsen, 2005 and references therein).
To define the essential roles for the MALS complex in mammals, the MALS complex was purified from brain. Isolation of the MALS complex revealed an association with a family of cytoskeletal and presynaptic adhesion molecules. Importantly, liprin-α1, -α2, -α3, and -α4 in the MALS complex. Association with this complex is mediated through the SAM domains in liprin-α and an NH2-terminal region in CASK. Using the sterile α motif (SAM) domains of liprin-α as a dominant negative, the MALS–liprin complex was disrupted in dissociated neurons. To understand the function of the MALS complex, mutant mice were generated lacking all three MALS genes. Mice lacking any single gene were viable and fertile. However, mice lacking all three MALS genes died within one hour of birth. This perinatal lethality is associated with impaired presynaptic function, reflecting the presynaptic deficits of invertebrates lacking liprin-α orthologues. These studies establish a crucial role for the MALS complex in synaptic vesicle exocytosis and implicate liprin-α in this process (Olsen, 2005).
Synaptogenesis is a highly regulated process that underlies formation of neural circuitry. Considerable work has demonstrated the capability of some adhesion molecules, such as SynCAM and Neurexins/Neuroligins, to induce synapse formation in vitro. Furthermore, Cdk5 gain of function results in an increased number of synapses in vivo. To gain a better understanding of how Cdk5 might promote synaptogenesis, potential crosstalk between Cdk5 and the cascade of events mediated by synapse-inducing proteins was investigated in a mammalian system. One protein recruited to developing terminals by SynCAM and Neurexins/Neuroligins is the MAGUK family member CASK. It was found that Cdk5 phosphorylates and regulates CASK distribution to membranes. In the absence of Cdk5-dependent phosphorylation, CASK is not recruited to developing synapses and thus fails to interact with essential presynaptic components. Functional consequences include alterations in calcium influx. Mechanistically, Cdk5 regulates the interaction between CASK and liprin-α. These results provide a molecular explanation of how Cdk5 can promote synaptogenesis (Samuels, 2007).
Homologs of liprin-α proteins are essential for presynaptic terminal formation in C. elegans and Drosophila . Mutations in C. elegans syd-2 result in a diffuse localization of several presynaptic proteins and abnormally sized active zones, and loss- and gain-of-function experiments demonstrate that presynaptic organization is dependent on syd-2. Likewise, Dliprin-α is required for normal synaptic morphology including the size and shape of the presynaptic active zone in Drosophila . Cdk5-dependent phosphorylation of CASK occurs in both the CaMK and L27 domains, and only mutation of both sites yields a localization phenotype. Since liprin-α proteins require the presence of both domains to interact with CASK, the phosphorylation sites are in a prime spot to mediate the interaction. According to the model described in this study, liprin-α is required for initial CASK localization to presynaptic terminals. Since, liprin-α binds directly to the kinesin motor KIF1A and in Drosophila liprin-α mutant axons there is decreased anterograde processivity resulting in reduced levels of presynaptic markers at terminals, it is feasible that liprin-α acts as a cargo receptor that delivers CASK, as well as other components, to and within the developing synapse. Cdk5-dependent phosphorylation could then act to coordinate distinct pools of CASK that are bound to liprin-α or are bound to other components of the presynaptic machinery. Importantly, it is not believed that Cdk5 loss of function generally affects liprin-α-mediated transport since synaptophysin, a marker of synaptic vesicles, is still properly localized within synaptosomes. In this model, there would be advantages of having locally enhanced Cdk5 activity within the presynaptic terminal relative to some other cellular compartments. Supporting this idea, phospho-CASK is particularly enriched at synaptic membranes, and Cdk5 has been shown to phosphorylate and regulate several proteins, including Munc-18, Dynamin-1, Amphiphysin-1, and Synaptojanin-1, that function to control multiple rounds of the synaptic vesicle cycle. Synapsin-1 is also a Cdk5 substrate. With regard to the role of liprin-α, it will ultimately be essential to assay synapse formation and CASK localization in mammalian liprin-α loss-of-function models (Samuels, 2007).
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