BIOLOGICAL OVERVIEW

Mutations in rho-type guanine exchange factor (rt/GEF), also called dpix, were recovered from a large-scale screen in Drosophila for genes that control synaptic structure. dPix/rtGEF is homologous to mammalian Pix (Werner, 1997). dPix plays a major role in regulating postsynaptic structure and protein localization at the Drosophila glutamatergic neuromuscular junction. dpix mutations lead to decreased synaptic levels of the PDZ protein Discs large, the cell adhesion molecule Fas II, and the glutamate receptor subunit GluRIIA, and to a complete reduction of the serine/threonine kinase Pak and the subsynaptic reticulum. The electrophysiology of these mutant synapses is nearly normal. Many, but not all, dpix defects are mediated through dPak, a member of the family of Cdc42/Rac1-activated kinases. Direct interaction of mammalian Pix with Pak has been detected. Thus, a Rho-type GEF (Pix) and Rho-type effector kinase (Pak) regulate postsynaptic structure (Parnas, 2001).

The synapse is a highly specialized structure composed of a presynaptic terminal and a postsynaptic region. Both sides contain specific molecules that are involved in the regulation and plasticity of synaptic transmission. Electron micrographs reveal a dense thickening below the postsynaptic membrane, called the postsynaptic density (PSD). The PSD contains a variety of neurotransmitter receptors, linker proteins, signal transduction proteins, cytoskeletal elements, cell adhesion molecules, ion channels, enzymes, and other regulatory components (Parnas, 2001).

Less is known about the signaling pathways that control the assembly and stabilization of these components. While biochemical approaches have been extremely successful in the identification of PSD components, such approaches often rely on the direct and stable interaction between proteins. Genetic screens offer a complementary approach toward identifying molecules based on their function, and have the potential to reveal key regulatory components that are upstream of the clustered protein complexes. The neuromuscular junction (NMJ) of Drosophila is ideal for such a genetic analysis. The Drosophila NMJ is a glutamatergic synapse composed of boutons similar in many respects to mammalian CNS synapses. A noninvasive method has been developed to image synaptic growth using a green fluorescent protein (GFP) chimeric protein marker that is transgenically expressed in muscle and that localizes specifically to the PSD. Using this GFP marker, a large-scale mutant screen was conducted in search of genes that control synapse formation, synaptic structure, and synaptic growth. Genes were uncovered that control important aspects of all of these aspects of the synapse. dpix (rtGEF) gene (Werner, 1997) regulates synaptic structure and protein localization at the Drosophila NMJ (Parnas, 2001).

In mammals, the Pix family contains two members: alphaPix (Cool-2) and ßPix (Cool-1) (Bagrodia, 1998; Manser, 1998). Pix has an SH3 domain, a DBL-homology GEF domain, and a pleckstrin homology domain. The Cool (for cloned-out of library)/Pix (for PAK-interactive exchange factor) proteins directly bind to members of the PAK family of serine/threonine kinases and regulate their activity. In Drosophila, dPix is localized to the PSD: dpix mutations lead to the loss of synaptic Pak kinase. Paks are a family of Cdc42/Rac1-activated serine/threonine kinases important in regulating actin-containing structures. In the fly NMJ, Pak kinase is localized to the PSD. In mammals, Pak is recruited to focal complexes in a Cdc42-, Rac1-, and Pix- dependent manner (Parnas, 2001).

In addition to the absence of Pak kinase at the synapse, dpix mutations lead to the decrease in synaptic levels of the PDZ protein Discs-large (Dlg), the cell adhesion molecule Fasciclin II (Fas II), the glutamate receptor (GluR) subunit GluRIIA, and to the elimination of the subsynaptic reticulum (SSR). In Drosophila, the PSD-95 homolog Dlg has been shown to be directly responsible for the clustering of the Shaker potassium channel and to partially control the clustering of the cell adhesion molecule Fas II to the NMJ. Many, but not all, dpix defects are mediated through Pak kinase. Thus, the data suggest a pathway for synaptic clustering from dPix to Pak kinase to Dlg to Shaker and to Fas II (Parnas, 2001).

Previous biochemical studies on mammalian glutamatergic synapses have shown that several regulators, or effectors of Ras and Rho-type GTPases, are enriched at the PSD and bind to PSD-95, including SynGAP, and Kalirin. GEFs were also implicated in the organization of the synapse; Collybistin plays a role in the in vitro clustering of glycine and GABA receptors. Rho-type GTPases are also implicated in the in vitro clustering of neurotransmitter receptors, for example, at the cholinergic NMJ in vertebrates. Rho-type GTPases are known to play a major role in the regulation of the actin cytoskeleton, and thus they are good candidates to control the PSD. The data described here show an important role in synapse organization for a Rho-type GEF and a Rho-type effector kinase, and suggest a functional role for Rho-type GTPases in the regulation of postsynaptic structure and protein localization. However, it is not yet known which Rho-type GTPase is involved (e.g., Cdc42 vs. Rac). Alternatively, it is possible that the localization of the Pix-Pak complex to the synapse is independent of small G proteins. There is a precedent for Pak and/or Pix acting independently of Cdc42 and Rac, and so it is possible that small G proteins are not necessary to localize Pix/Pak to the synapse (Parnas, 2001 and references therein).

The dpix phenotype is consistent with at least two functions at the postsynaptic compartment: targeting and stabilization of postsynaptic components. In dpix mutants, Pak kinase is completely missing from the synapse. Since Pix is known to directly interact with Pak in mammals and target it to focal complexes (Manser, 1998), the data best fit with the model in which dPix targets Pak kinase to the synapse via a direct interaction. Furthermore, overexpressing either Pak kinase or a membrane-tethered gain-of-function form of Pak kinase does not result in any accumulation of Pak kinase at the synapse. Still, it is possible that Pak kinase is targeted to the synapse via a different mechanism and fails to stabilize in dpix mutants (Parnas, 2001).

In contrast to Pak kinase, Dlg and GluRIIA are not completely eliminated from the synapse in dpix mutants, but rather, their levels are reduced. In the case of Dlg, its localization pattern is also disrupted, indicating that dPix controls the postsynaptic targeting of Dlg at least to some extent, as well as its stabilization at the synapse. The localization pattern of GluRIIA (in subsynaptic domains opposite active zones) is intact. Thus, dPix is not necessary for the synaptic targeting of GluRIIA per se, but rather, it is important for maintenance of its levels and/or stabilization (Parnas, 2001).

dPix could actively control the stabilization of Dlg and GluRIIA (presumably through Pak kinase) or indirectly through the loss of the SSR. The latter explanation is not favored for several reasons. (1) The dpix phenotype is already evident in early first instar larvae when an elaborate SSR has not yet been formed and so any defect in the localization of synaptic components cannot be attributed to the absence of SSR. (2) In vertebrates as well as in flies, the neurotransmitter receptors are actually clustered close to the synaptic cleft rather than in the depth of the secondary folds or the SSR. Therefore, GluRIIA should not be dramatically affected by the reduction of the SSR. (3) Preliminary evidence shows that dpix is able to suppress the phenotype of another mutant isolated in the screen. In this mutant, Dlg is clustered at the synapse but also in extra-synaptic sites, which would not contain the SSR, indicating that dPix is involved primarily in targeting or stabilizing Dlg, rather than in formation of the SSR (Parnas, 2001).

How does dPix function to stabilize Dlg and GluRIIA? A major downstream effector of dPix at the synapse is Pak kinase. In dpak mutants, the levels of Dlg and GluRIIA at the synapse are reduced, and the SSR is also disrupted. dPix seems to have other partners apart from Pak kinase: (1) the levels of GluRIIA at the synapse are more severely affected in dpix, than in dpak mutants; (2) in dpix mutants, both synaptic Dlg levels and its localization pattern, are compromised, whereas in dpak mutants, the localization pattern of Dlg is unaffected; (3) the SSR is more severely compromised in dpix than in dpak mutants. Therefore, whereas Pak kinase is the major effector of dPix in stabilizing Dlg and GluRIIA at the synapse, it seems that there are other dPix binding partners that also affect Dlg targeting and/or clustering, as well as SSR formation (Parnas, 2001).

One way in which Pak could control the stabilization of postsynaptic components is by controlling the actin cytoskeleton. Pak is known to regulate actin dynamics. The actin cytoskeleton is important for the localization of various synaptic components, including the AChR, GluRs, the GlyR associated protein gephyrin, and CaMKIIalpha (Parnas, 2001 and references therein).

Pak is known to be an effector of Rac and Cdc42, and mammalian Pix has been shown to activate Rac (Manser, 1998). It was therefore examined whether cdc42 mutants or dRac1 dominant-negative or constitutively active transgenes have any effect on the localization of postsynaptic proteins. No postsynaptic defects were detected either in synaptic protein levels or in SSR structure in these flies. All of the G protein alleles or transgenic flies used were hypomorphs. It is possible that a phenotype was not observed because only a limited number of reagents are available, and none of the flies that survived to larval stage represented the complete loss-of-function condition (Parnas, 2001).

The localization of dPix, as assessed by antibody staining, is mainly postsynaptic although motorneurons are stained at lower levels. Moreover, there are clearly presynaptic defects in dpix mutants, including structural and electrophysiological defects. It seems likely that dPix is localized both pre- and post-synaptically, much like Dlg and Fas II. On the postsynaptic side, dPix is localized to active zones, along with Pak kinase and GluRII. Its absence, however, affects both active zone components (i.e., Pak kinase and GluRIIA) and periactive zone components, such as Dlg. Another periactive zone component, Fas II, is not as affected by the absence of dPix. This is not a surprising result, however, since in the absence of Dlg, a large proportion of Fas II is still correctly localized to the synapse. In dpix mutants, Dlg is reduced by approximately 80%, but it is not completely absent from the synapse, so the effect on Fas II is expected to be even less pronounced. How may dPix affect periactive zones? The most likely explanation at the moment is that dPix localizes Pak kinase to postsynaptic active zones and that the periactive zones are then constructed by a cytoskeletal meshwork that emanates around these active zones. At the presynaptic terminal, dPix seems to act via a different cascade: (1) Pak kinase is not concentrated at the presynaptic terminal; (2) dPix does not affect presynaptic Dlg, since the electrophysiological defects associated with the absence of presynaptic Dlg are not seen (Parnas, 2001).

The SSR may be viewed as analogous to the junctional-folds at the vertebrate NMJ. What may be the function of the SSR in flies or the junctional-folds in vertebrates? The main postsynaptic electrophysiological defects found in dpix mutants are ~15% reduction in both mEJPs and EJPs. The reduction in mEJPs is most likely due to the reduction in the levels of GluRIIA. However, in GluRIIA nulls there is no decrease in EJP amplitude due to a retrograde signal regulating presynaptic transmitter release. As a result, in GluRIIA nulls there is a large increase in quantal content, whereas in dpix mutants, quantal content remains unchanged. Although the changes in mEJP and EJP amplitudes are relatively small, this data could be interpreted to mean that in dpix mutants the retrograde signal is compromised. This possibility is being investigated further by looking at dpix and DGluRIIA double mutants. How could the retrograde signal be disrupted? In vertebrates, there is a spatial segregation of synaptic components due to the existence of junctional-folds. In the fly there is no extensive evidence for segregated localization of postsynaptic components in different regions of the SSR. However Dlg is more widely distributed than the GluRII receptor. This segregation of synaptic components could potentially be important for the segregation of different signaling cascades that are important for the generation of the retrograde signal, and presumably for other, as yet unidentified, synaptic functions. In dpix mutants, both the levels of various synaptic components, as well as the integrity of the periactive zone are affected, possibly leading to the disruption of various signaling cascades (Parnas, 2001).

Another possible function for the fly SSR is that it functions as a regional endoplasmic reticulum (ER). In vertebrate muscles, only the nucleus that is close to the synapse is active in transcribing mRNAs of synaptic proteins. The nuclei in fly muscles have no such specialization. The SSR could thus serve as a specialized region for the production of synaptic components. Indeed the SSR has been shown to be a site of translation of GluRIIA. The notion of a nonclassical ER like function for the SSR is supported also from vertebrate studies. In rat spinal cord, there are specialized RER-like cisternae in dendritic spines. In this way, it could be that the relatively simple junctional-folds of vertebrates are expanded in the fly SSR to serve a second function for local protein synthesis that in vertebrates is handled by localized nuclei on the postsynaptic side of the NMJ (Parnas, 2001).

GENE STRUCTURE

cDNA clone length - 3362

Bases in 5' UTR - 501

Exons - 8

Bases in 3' UTR - 797

PROTEIN STRUCTURE

Amino Acids - 687

Structural Domains

A Drosophila gene, rho-type guanine exchange factor (rtGEF) has been identified that has substantial sequence homology to a distinct class of vertebrate proto-oncogenes that includes DBL, VAV, Tiam-1, ost and ect-2. It has predicted Rho or Rac guanine exchange factor (Rho/RacGEF) and pleckstin homology (PH) domains with the PH immediately downstream of the Rho/RacGEF. Rho/RacGEFs catalyze the dissociation of GDP from the Rho/Rac subfamily of Ras-like GTPases, thus activating the target Rho/Rac. Members of the Rho/Rac subfamily regulate organization of the actin cytoskeleton, which controls the morphology, adhesion and motility of cells (Werner, 1997).

RtGEF is most homologous to the vertebrate protein ßPix. Pix was initially identified as a protein that binds Pak through its SH3 domain (Bagrodia, 1998; Manser, 1998; Parnas, 2001).

date revised: 1 June 2024

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