nwk (nervous wreck), a temperature-sensitive paralytic mutant, causes excessive growth of larval neuromuscular junctions (NMJs), resulting in increased synaptic bouton number and branch formation. Ultrastructurally, mutant boutons have reduced size and fewer active zones, associated with a reduction in synaptic transmission. nwk encodes an FCH and SH3 domain-containing adaptor protein that localizes to the periactive zone of presynaptic terminals and binds to the Drosophila ortholog of Wasp (Wsp), a key regulator of actin polymerization. wsp null mutants display synaptic overgrowth similar to nwk and enhance the nwk morphological phenotype in a dose-dependent manner. Evolutionarily, Nwk belongs to a previously undescribed family of adaptor proteins that includes the human srGAPs, which regulate Rho activity downstream of Robo receptors. It is proposed that Nwk controls synapse morphology by regulating actin dynamics downstream of growth signals in presynaptic terminals (Coyle, 2004).

SH3 adaptor proteins, characterized by the presence of SRC homology 3 (SH3) domains, commonly link transmembrane signaling molecules with cytoskeleton-associated effectors in the cytoplasm. They play a major role in axon pathfinding by recruiting proteins that affect actin polymerization downstream of guidance cue receptors to generate acute changes in growth cone movement. For example, Dreadlocks (Dock), which contains three SH3 domains, facilitates Slit-Robo repulsion in commissural axons of the Drosophila CNS and also mediates projection of retinal axons into optic lobes. Dock recruits proteins that affect Rho GTPase activation, as well as the Rho-dependent kinase, Pak, downstream of transmembrane receptors such as Robo and Dscam (Coyle, 2004 and references therein).

In organisms ranging from yeast to humans, cytoskeletal growth is controlled by the Rho GTPases, including RhoA, Rac, and Cdc42. They act as binary switches that are active in the GTP bound form and inactive in the GDP bound form. Rho signaling is modulated by GTPase activating proteins (GAPs) that accelerate hydrolysis of GTP and guanine nucleotide exchange factors (GEFs) that promote GTP for GDP exchange. Rho proteins and their effectors are highly expressed in nerve terminals and are known to regulate neurite extension, guidance, branching, and stability. Genetic analysis in Drosophila has demonstrated roles for Rho effectors in synapse formation. For example, loss-of-function mutations in p190RhoGAP and the RhoGEF, still life (sif), affect axon growth in mushroom body neurons and motor neurons, respectively. Also, the RhoGEF Trio combines with Dock and Pak to activate Rac during photoreceptor axon guidance (Coyle, 2004 and references therein).

Activated Rho proteins can activate members of the Wiscott-Aldrich Syndrome Protein (Wasp) family. Wasp and its homologs, N-Wasp and the Wave/Scar proteins, promote F-actin assembly at the leading edge of motile cells by activating the ARP2/3 (actin-related protein) complex. This regulation is essential for the formation of lamellipodia and filopodia during axonal and dendritic growth in mammalian neurons. Wasp and N-Wasp contain a CRIB/PBD domain that binds activated Cdc42, an interaction that is required for efficient stimulation of ARP2/3. The Wave/Scars do not directly bind Rho GTPases, but can be activated through binding to Rac-associated intermediaries like IRSp53. All Wasp/N-Wasp and Wave/Scar relatives contain a central proline-rich domain that is recognized by SH3 adaptor proteins in numerous signal transduction pathways. Including Dock and its mammalian homolog, Nck, these SH3 adaptors control the spatial and temporal activation of the Wasps (Coyle, 2004 and references therein).

In addition to growth cone motility, Wasp-dependent F-actin polymerization also plays a role in some types of vesicle transport and endocytosis. Although Wasp and its homologs are not yet implicated in synaptic transmission, functional analysis has revealed a requirement for F-actin in synaptic vesicle recycling and for the induction of long-term potentiation, during which rapid formation and retraction of actin-rich filopodia and dendritic spines are observed both pre- and post-synaptically. Despite the pervasive role of actin in neuronal growth and function, the molecular pathways involving Rho and Wasp at synapses are not well understood (Coyle, 2004).

Nwk, a novel synaptic adaptor protein, binds the Drosophila ortholog of Wasp (Wsp) and regulates both NMJ growth and activity. Nwk has a distinct domain organization, including an N-terminal FCH and two SH3 domains, that is conserved throughout evolution. Mutation of a human Nwk homolog, srGAP3 (Slit-Robo GTPase Activating Protein), is associated with severe mental retardation (Endris, 2002), suggesting that Nwk proteins regulate the development of both vertebrate and invertebrate nervous systems (Coyle, 2004).

Thus nwk encodes an SH3 adaptor protein that interacts with Wsp, a principal regulator of ARP2/3-dependent actin polymerization. Regulated actin polymerization via WASP and its homologs is thought to underlie neurite extension, cell junction formation, receptor-mediated endocytosis, and other processes relevant to synaptic growth and function. These results reveal a novel molecular pathway that potentially links signal transduction and actin dynamics in synapses (Coyle, 2004).

Nwk negatively regulates larval NMJ growth, since null mutants exhibit increased bouton number, branch formation, and total length. In addition, nwk adults undergo rapid temperature-sensitive (ts) paralysis at 38°C, suggesting that Nwk continues to have an important function(s) in the adult nervous system that may include a direct role in neurotransmission. The molecular mechanism of ts paralysis is not understood at present, and further studies are underway to determine how this behavioral defect is related to the morphological changes in nwk synapses. Nevertheless, it has been verified by several means that all of these phenotypes arise from mutations of the same gene. (1) The same abnormalities are observed when nwk is homozygous as when it is hemizygous over Df(3L)Rdl2. (2) Multiple independent alleles of nwk isolated by failure to complement the adult ts paralytic phenotype all display identical changes in larval NMJ development. (3) Each of these phenotypes is partially or completely rescued by panneural expression of a transgene that contains a complete nwk ORF (Coyle, 2004).

While panneural expression of Nwk rescues both larval and adult phenotypes, expression in muscle does not rescue either, indicating that Nwk does not function postsynaptically. In agreement with these observations, it was found that Nwk expression in wild-type larvae is restricted to neurons and is localized to a distinct presynaptic, subcellular region of synaptic boutons termed the periactive zone. It has been proposed that the periactive zone is specialized for regulating synaptic development. It encompasses the area immediately surrounding synaptic densities and contains several known regulators of synaptic growth and plasticity, including the cell adhesion molecule, FasII, the RhoGEF, Sif, and the microtubule-associated protein, DVap-33A. Thus, Nwk is positioned at a critical juncture between transmembrane receptors, cytoskeletal proteins, and active zones (Coyle, 2004).

Nwk contains an N-terminal FCH domain, a novel ARNEY domain, two SH3 domains, and many polyproline sequences. This particular domain arrangement is distinct, found only in Nwk and 11 other homologs in organisms ranging from yeast to humans. Therefore, Nwk is a member of a small subfamily of evolutionarily conserved SH3 adaptor proteins that includes other potential regulators of synaptic development and function. The only substantial difference in domain structure among these 12 proteins is that 7 of them have a RhoGAP domain in place of the SH3a domain. Among the six human or three mouse Nwk homologs, there are proteins of both structural types. In contrast, the D. melanogaster and S. cerevisiae genomes each contain a single Nwk ortholog lacking the RhoGAP domain but containing a second SH3 domain. Nevertheless, the Nwk to Rho signaling pathway may be conserved in these organisms because the S. cerevisiae ortholog of Nwk, Bzz1p, was found to coprecipitate with the RhoGAP protein, ECM25 (Gavin, 2002). Similar to Dock, Nwk is likely to interact with multiple targets in a variety of cellular contexts by virtue of its modular protein binding domains (Coyle, 2004).

It is proposed that Nwk and Wsp interact directly as components of a signal transduction pathway in neurons controlling NMJ growth and bouton branching on the basis of several lines of evidence. (1) The SH3a domain of Nwk binds Wsp in vitro, and Wsp interacts directly with the SH3 domains of Nwk in the yeast two-hybrid system. (2) Nwk and Wsp immunoreactivity overlap in discrete regions of periactive zones. (3) nwk and wsp null mutations increase synaptic growth and bouton branching in identical fashion. (4) Each mutant can be rescued by presynaptic expression of the respective wild-type gene. (5) The severity of the nwk and wsp mutant phenotypes are enhanced by removal of the other gene in a dose-dependent manner. (6) The proposed interaction between Nwk and Wsp is supported by studies in yeast, demonstrating that the ortholog of Nwk, Bzz1p, binds to the ortholog of Wasp, Las17p, promoting actin polymerization and regulating yeast cell growth (Soulard, 2002; Coyle, 2004 and references therein).

Wasp and its relatives such as Wave/Scar stimulate the ARP2/3 complex, which nucleates and crosslinks F-actin. Other neuronally expressed proteins have distinct effects on the actin cytoskeleton. For example, Cortactin stablizes F-actin branches and promotes ARP2/3 activity independently of Wasp; Ena/Vasp increases the rate of actin filament extension; and Cofilin severs existing filaments to accelerate F-actin turnover. The Drosophila genome contains homologs of scar, cortactin, ena, cofilin, and others that may function either in parallel or antagonistically to wsp. Consequently, loss of Wsp may only partially or selectively disrupt F-actin polymerization at the synapse. In support of this possibility, phalloidin staining was not abolished in wsp mutant NMJs, although the precise arrangement of individual actin filaments cannot be resolved (Coyle, 2004).

Adaptor proteins such as Nwk may specify which combinations of actin regulators are recruited in response to specific cues. Different combinations of actin regulators may contribute to distinct cellular mechanisms such as adhesion, axon extension, endocytosis, and vesicle transport. It is proposed that the morphological abnormalities observed in nwk and wsp single and double mutants result from altered activity in a Nwk/Wsp/ARP2/3-dependent pathway that regulates one or more of these mechanisms within periactive zones (Coyle, 2004).

The involvement of ARP2/3 is inferred from the extensively documented role of Wsp in various cell types including neurons. However, the possibility cannot be excluded that the Nwk/Wasp interaction in boutons may involve other, novel functions (Coyle, 2004).

Recent analysis of three human Nwk homologs, the Slit-Robo GAPs (srGAPs 1-3), supports the proposal that Nwk family members link transmembrane signals with cytoskeletal effectors. Robo receptors bind secreted ligands, Slits, to control neuronal migration and axon guidance in mammals and insects. The srGAPs share the same domain organization with Nwk, except they contain one SH3 domain and a RhoGAP domain instead of two SH3 domains. The SH3 domain of the srGAPs binds to the cytoplasmic CC3 domain of activated Robo1, and the neighboring RhoGAP domain locally inactivates Cdc42 (Wong, 2001). As a result, ARP2/3-dependent F-actin assembly is decreased downstream of activated Robo and migrating neurons are repulsed from Slit. This activity is important for CNS development, since mutations of srGAP3 are associated with severe mental retardation (Endris, 2002). Thus, Nwk and its relatives potentially regulate F-actin polymerization and synaptic growth in both vertebrates and invertebrates (Coyle, 2004).

In Drosophila, mutations in nwk lead not only to increased NMJ growth, but also to decreased synaptic transmission. At nwk NMJs, quantal content is reduced by more than half, indicating that presynaptic vesicle release is curtailed. However, it is not believed that the overgrowth observed in nwk NMJs is a secondary, compensatory effect of reduced synaptic transmission for several reasons: (1) mutations that directly decrease quantal content at larval NMJs, such as in synaptotagmin and methuselah, do not cause compensatory overgrowth in presynaptic terminals; (2) Nwk localizes to the periactive zone and is therefore unlikely to have a primary effect on synaptic vesicle exocytosis in active zones; (3) no decreases were detected in the distribution or size of synaptic vesicles in the ultrastructural analysis of synaptic boutons or in functional assays of quantal size; (4) normal endocytosis of synaptic vesicles was observed in nwk. These results indicate that Nwk is not directly involved with synaptic vesicle dynamics. Instead, ultrastructural analysis shows that synaptic contacts are fewer and smaller in nwk boutons: this may account for the reduced synaptic currents. Future experiments will address whether this phenotype is also mediated via Wsp or through other effectors (Coyle, 2004).

PROTEIN STRUCTURE

Amino Acids - 1001

Structural Domains

nwk was identified by positional cloning. Recombination mapping placed nwk 1 map unit to the right of hairy (3-26.5); this corresponds approximately to cytological interval 66E-67A. Therefore, Df(3L)29A6,, which is deleted for 66F5-67B1, was tested; it failed to complement the ts paralysis of nwk. Df(3L)Rdl2, which contains a small deletion within 66F5 that includes the GABA-A receptor gene Rdl, also uncovered nwk. Complementation tests indicated that nwk and Rdl null mutants are not allelic (Coyle, 2004).

The breakpoints of Df(3L)Rdl2 were mapped by Southern blot analysis using overlapping cosmids that spanned 66F. The Drosophila genome database lists 20 ORFs within this interval, representing candidates for the nwk locus. Sequence analysis revealed that one of these candidates, CG4684, a predicted ORF with homology to Dock, contained a unique polymorphism in each of three alleles (nwk^2,4,5). The remaining alleles, nwk¹ and nwk³, had molecular lesions downstream of CG4684 but within an overlapping EST (SD0455), suggesting that the predicted ORF in the database was incomplete. Therefore, the 5′ end of CG4684 was isolated via RACE and this sequence was used to probe an adult Drosophila cDNA library. Several cDNAs were recovedred that linked CG4684 and SD04555 into a single full-length ORF encoding a 1001 amino acid protein. All five nwk alleles contained unique molecular lesions within this ORF (Coyle, 2004).

nwk encodes a predicted 110 kDa protein with several recognizable functional domains. An Fes/CIP4 Homology (FCH) domain is present at the N terminus. The function of this domain is unknown, but it has been implicated in actin binding. Following the FCH domain are two canonical SH3 domains, modular protein binding domains that recognize proline-rich consensus sequences, often found in signal transduction pathways linking the membrane and cytoskeleton. In addition, the C terminus of Nwk contains a proline-rich segment with at least five potential SH3 binding sites (Coyle, 2004).

Database searches identified 11 related proteins in yeast, worms, and mammals, including the three human srGAPs. Nwk and these 11 homologs share a highly conserved 60 amino acid sequence adjacent to the FCH domain that is not present in any other proteins. This is referred to as the ARNEY domain based on its central consensus sequence ARNEYLL. There are six Nwk relatives in humans and three in mice. Drosophila, C. elegans, and S. cerevisiae each contain a single Nwk protein. Seven of the twelve Nwk family members differ in that the first SH3 domain (SH3a) is replaced by a RhoGAP domain. The presence of FCH, ARNEY, RhoGAP, and SH3 domains suggests that Nwk and its homologs compose a distinct family of adaptor proteins that interact with cytoskeletal regulators (Coyle, 2004).

EVOLUTIONARY HOMOLOGS

The Slit protein guides neuronal and leukocyte migration through the transmembrane receptor Roundabout (Robo). The intracellular domain of Robo interacts with a novel family of Rho GTPase activating proteins (GAPs). Two of the Slit-Robo GAPs (srGAPs) are expressed in regions responsive to Slit. Slit increases srGAP1-Robo1 interaction and inactivates Cdc42. A dominant negative srGAP1 blocks Slit inactivation of Cdc42 and Slit repulsion of migratory cells from the anterior subventricular zone (SVZa) of the forebrain. A constitutively active Cdc42 blocks the repulsive effect of Slit. These results have demonstrated important roles for GAPs and Cdc42 in neuronal migration. A signal transduction pathway is proposed, from the extracellular guidance cue to intracellular actin polymerization (Wong, 2001).

In Saccharomyces cerevisiae, the WASP (Wiskott-Aldrich syndrome protein) homolog Las17p (also called Bee1p) is an important component of cortical actin patches. Las17p is part of a high-molecular-weight protein complex that regulates Arp2/3 complex-dependent actin polymerization at the cell cortex and that includes the type I myosins Myo3p and Myo5p and verprolin (Vrp1p). To identify other factors implicated with this complex in actin regulation, proteins were isolated that bind to Las17p by two-hybrid screening and affinity chromatography. Lsb7/Bzz1p (for Las seventeen binding protein 7) is an Src homology 3 (SH3) domain protein that interacts directly with Las17p via a polyproline-SH3 interaction. Bzz1p coimmunoprecipitates in a complex with Las17p, Vrp1p, Myo3/5p, Bbc1p, Hsp70p, and actin. It colocalizes with cortical actin patches and with Las17p. This localization is dependent on Las17p, but not on F-actin. Bzz1p interacts physically and genetically with type I myosins. While deletion of BZZ1 shows no obvious phenotype, simultaneous deletion of the BZZ1, MYO3, and MYO5 genes is lethal. Overexpression of Bzz1p inhibits cell growth, and a bzz1Delta myo5Delta double mutant is unable to restore actin polarity after NaCl stress. Finally, Bzz1p in vitro is able to recruit a functional actin polymerization machinery through its SH3 domains. Its interactions with Las17p, Vrp1p, and the type I myosins are essential for this process. This suggests that Bzz1p could be implicated in the regulation of actin polymerization (Soulard, 2002).

In the last few years, several genes involved in X-specific mental retardation (MR) have been identified by using genetic analysis. Although it is likely that additional genes responsible for idiopathic MR are also localized on the autosomes, cloning and characterization of such genes have been elusive so far. A previously uncharacterized gene, MEGAP, has been isolated that is disrupted and functionally inactivated by a translocation breakpoint in a patient who shares some characteristic clinical features, such as hypotonia and severe MR, with the 3p(-) syndrome. By fluorescence in situ hybridization and loss of heterozygosity analysis, it has been demonstrated that this gene resides on chromosome 3p25 and is deleted in 3p(-) patients that present MR. MEGAP/srGAP3 mRNA is predominantly and highly expressed in fetal and adult brain, specifically in the neurons of the hippocampus and cortex, structures known to play a pivotal role in higher cognitive function, learning, and memory. Several MEGAP/srGAP3 transcript isoforms are described; MEGAP/srGAP3a and -b represent functional GTPase-activating proteins (GAP) by an in vitro GAP assay. MEGAP/srGAP3 has recently been shown to be part of the Slit-Robo pathway regulating neuronal migration and axonal branching, highlighting the important role of MEGAP/srGAP3 in mental development. It is proposed that haploinsufficiency of MEGAP/srGAP3 leads to the abnormal development of neuronal structures that are important for normal cognitive function (Endris, 2002).

date revised: 23 June 2004

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